Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
SYSTEM AND METHOD FOR TARGET MATERIAL RETRIEVAL FROM
MI CROWELLS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application number
62/866,726 filed on 26-JUN-2019, which is incorporated in its entirety herein
by this
reference.
[0002] This application also claims the benefit of U.S. Application
number 16/867,235
filed 05-MAY-2020, which claims the benefit of U.S. Provisional Application
number
62/844,470 filed 07-MAY-2019, which are each incorporated in its entirety
herein by this
reference.
TECHNICAL FIELD
[0003] This invention relates generally to the cell capture and cell
processing field, and
more specifically to a new and useful system and method for target material
retrieval in the
cell capture and cell processing field.
BACKGROUND
[0004] With an increased interest in cell-specific drug testing,
diagnosis, and other
assays, systems and methods that allow for individual cell isolation,
identification, and
retrieval are becoming highly desirable. Single cell capture systems and
methods have been
shown to be particularly advantageous for these applications. However,
associated processes
and protocols for single cell capture and subsequent analysis must often be
performed in a
particular manner and with a high precision in order to properly maintain the
cells.
Furthermore, efficient retrieval of target material from high density
platforms is subject to
many challenges. As such, these processes can be time consuming for the user,
require
extensive and iterative manual library preparation and selection processes,
not amenable to
automation as well as result in damage to the cells or otherwise unfavorable
results if they are
not performed properly.
1
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0005] Thus, there is a need in the cell capture and cell processing
field to create a new
and useful system and method for sample processing and target material
retrieval and
minimize steps required in the library preparation of the target biomaterials.
BRIEF DESCRIPTION OF THE FIGURES
[0006] FIGURE iA depicts a schematic of an embodiment of a system for
target
material retrieval.
[0007] FIGURE 1B depicts schematics of applications of an embodiment of a
system
for target material retrieval.
[0008] FIGURES 2A-2C depict views of a variation of a sample processing
cartridge
associated with a system for target material processing and retrieval.
[0009] FIGURES 3A-3C depict operation modes of a lid-opening tool
associated with
the sample processing cartridge shown in FIGURES 2A-2C.
[0010] FIGURES 4A-4B depict operation modes of a valve subsystem
associated with
the sample processing cartridge shown in FIGURES 2A-2C
[0011] FIGURE 5 depicts a variation of a process container for target
material
processing, retrieval, and downstream processing.
[0012] FIGURES 6A-6B depict schematic representations of an embodiment of
a
system for automated single cell sample processing.
[0013] FIGURES 7A-7C depict views of a variation of the system shown in
FIGURES
6A-6B;
[0014] FIGURES 8A-8C depict a first magnetic variation of a system for
target
material retrieval.
[0015] FIGURES 9A-9C depict a second magnetic variation of a system for
target
material retrieval.
[0016] FIGURES loA-loB depict variations of a subset of components used
for
material separation in a system for target material retrieval.
[0017] FIGURES 11A-11J depict operation modes of a separation subsystem
associated with a system for target material retrieval and processing;
2
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0018] FIGURES 12A-12D depict views of components of a variation of a
separation
subsystem associated with a system for target material retrieval.
[0019] FIGURES 13A-31C depict a second variation of a system for target
material
retrieval.
[0020] FIGURE 14 depicts a flowchart of an embodiment of a method for
target
material retrieval.
[0021] FIGURES 15A and 15B depict schematics of a variation of a method
for target
material retrieval.
[0022] FIGURE 16 depicts a flowchart of a method for target material
retrieval.
[0023] FIGURES 17A-17D depict schematics of a first example of a method
for target
material retrieval.
[0024] FIGURES 18A-18E depict a schematic of a second example of a method
for
target material retrieval.
[0025] FIGURE 19 depicts a flow chart of a variation of a method for
target material
retrieval.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0026] The following description of the preferred embodiments of the
invention is not
intended to limit the invention to these preferred embodiments, but rather to
enable any
person skilled in the art to make and use this invention.
1. Benefits
[0027] The system(s) and method(s) described can confer several benefits
over
conventional systems and methods.
[0028] The invention(s) confer(s) the benefit of providing mechanisms for
efficient
retrieval of target material (e.g., beads, cells, released nucleic acid
material, etc.) from high-
aspect ratio wells of a high-density capture platform. Retrieval is typically
difficult and non-
efficient in this scenario due to close packing of wells of the capture
platform. Retrieval
mechanisms described also subject target material to acceptable amounts of
shear and other
potential stresses that would otherwise obstruct downstream processing steps.
3
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0029] The invention(s) also confer(s) the benefit of reducing burden on
system
operators in relation to target material retrieval processes from wells, where
standard
processes can require repeating aspiration and dispensing steps that require
additional time.
[0030] The invention(s) can also confer the benefit of increasing the
efficiency at which
target material is retrieved (and non-target material is not retrieved).
Selective retrieval
efficiency can thus reduce downstream costs in relation to processing reagent
and other
material costs (due to reduced volumes needed) and processing burden. For
instance, the
invention(s) can enable a system operator to purchase smaller volumes of
reagents, reduce
the number of splits required for successful amplification of target molecules
and obviate the
need for doing SPRI-based clean-up and size selection of target
oligonucleotide products from
other oligonucleotide tags that do not contain products, but get carried over
from one process
step to the next. Such improved recovery of target products and reduction of
carryover of non-
target products can also reduce the complexity of data analysis and also
provide more useable
data pertaining to the desired biomarker analysis as well. This can function
to save costs,
reduce reagent waste, or have any other suitable outcome.
[0031] The invention(s) also confer the benefit of enabling at least
partial automation
of the protocols involved in single cell capture, target material retrieval,
and subsequent
processing. For instance, a human operator user can be removed from part or
all of the
method, in relation to protocols involving repeated purification, washing, and
retrieval steps.
Furthermore, the system(s) and/or method(s) can enable better accuracy in
performance of
a protocol over conventional systems and methods. Some of these inventions are
also much
amenable to full automation with a liquid handling robot.
[0032] Additionally or alternatively, the system and/or method can confer
any other
suitable benefit.
2. System
[0033] As shown in FIGURE rA, embodiments of a system 100 for target
material
retrieval include: an adaptor 110 configured to interface with/communicate
with a capture
region of a sample processing chip 132 for capturing particles in single-
particle format within
a set of wells, wherein the adaptor 110 can include a cavity 120 configured
to, in response to
an applied force, promote release of target material from the set of wells and
into or to the
adaptor 110 for efficient retrieval of target material from the chip 101.
Applied forces are
4
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
preferentially applied perpendicular (900 30 ) to the plane of the
microwells. Embodiments
of the system loo function to provide mechanisms for efficient retrieval of
target material
from a high-density capture device (e.g., microwell chip), where the high-
density capture
device includes a high-density array of high-aspect ratio microwells, in order
to promote
increased efficiency in captured single cell-bead pairing efficiency.
Embodiments of the
system loo can also function to reduce manual burden in relation to retrieval
of target
material from the high-density capture device. Embodiments of the system loo
can also
function to increase the efficiency at which target material is retrieved from
the high-density
capture device, and the efficiency at which non-target material is retained at
the capture
device.
[0034] In an embodiment, as shown in FIGURE 0 (top), a microwell of the
high-
density capture device can co-capture a functional particle with a single
biological cell having
target material. Then, the system can enable cell lysis and transfer of target
materials to the
functional particle within the microwell, and the system can enable
performance of reverse
transcription or ligation to link the target materials to an oligonucleotide
tag on the functional
particle. Then, the target material (and, in some embodiments, the functional
particle) can be
retrieved from the microwell. A specific example of the embodiment is shown in
FIGURE 0
(bottom), which depicts a top-down view of a high-density capture platform
having an array
of microwells, where some microwells contain a functional particle, some
microwells contain
a single cell, and some microwells are empty.
[0035] In relation to retrieval of target material in response to being
subject to one or
more applied forces, the applied forces can include one or more of: a magnetic
force; a gravity-
associated force (e.g., centrifugal force, buoyancy force, etc.); a fluid
pressure-driven force
produced by applying positive and/or negative pressure at the capture region
(e.g., through
an inlet channel of the chip 101, through an outlet channel of the chip um,
through an adaptor
manifold coupled to the chip 101, etc.); an electric field-associated force
(e.g., due to applied
voltage); ultrasound force; acoustic force; photo-generated pressure force;
laser-generated
shock force and any other suitable force.
[0036] In an embodiment of a fluid pressure-driven mechanism, the system
loo can
enable retrieval of target material from capture wells of the chip 101 with an
adaptor no
comprising structures for interacting with a pipettor, in a position where the
aspirator is
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
fluidly coupled to a fluid volume interfacing with the capture wells, and
where the system loo
includes a first operation mode for dispensing fluid into the fluid volume and
a second
operation mode for aspirating fluid from the fluid volume. The first operation
mode produces
local convective forces at the capture wells for loosening and lifting
material within the
capture wells, and the second operation mode produces convective currents for
delivery
material from the capture wells into the aspirator, where target material can
then be delivered
to an elution container. The system loo cycles between the first and the
second operation
modes to increase efficiency of target material from the capture wells.
Variations of this
embodiment can produce retrieved target material within 10-15 minutes of
manual operation
time, with a retrieval efficiency of 85-90% (in relation to percent of
captured particles that
are retrieved). Embodiments, variations, and examples of a fluid pressure-
driven system and
method are described in more detail in U.S. Application Number 15/815,532
titled "System
and Method for Retrieving and Analyzing Particles" and filed on 16-NOV-2017,
which is
incorporated in its entirety herein by this reference.
[0037] Other embodiments, variations, and examples of systems associated
with other
forces are described in more detail in Sections 2.2 and 2.3 below.
Furthermore, embodiments,
variations, and examples of the system can be configured to implement
embodiments,
variations, and examples of the method(s) described in Section 3 below.
[0038] In relation to sample processing, embodiments of the system loo
can include
or be configured to process cells, cell-derived material, and/or other
biological material (e.g.,
cell-free nucleic acids). The cells can include any or all of mammalian cells
(e.g., human cells,
mouse cells, etc.), embryos, stem cells, plant cells, microbes (e.g.,
bacteria, virus, fungi, etc.)
or any other suitable kind of cells. The cells can contain target material
(e.g., target ysate,
mRNA, RNA, DNA, proteins, glycans, metabolites, etc.) which originates within
the cells and
is optionally captured by the cell capture system for processing.
Additionally, the containers
containing the cells can be prepared from multiple cell-containing samples
(e.g., 12 samples,
24 samples, 48 samples, 96 samples, 384 samples, 1536 samples, other numbers
of samples),
wherein the various samples are hashed or barcoded prior to mixing them
together into a
single container (or reduced number of containers). Multiple samples may be
dispensed into
the same microwell chip by dispensing into geographically-distinct locations
of the chip. This
feature enables automated processing of multiple samples in the same automated
run for
6
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
their respective single cell preparation and library preparation operations.
Additionally or
alternatively, the system 100 can be configured to interact with particles
(e.g., beads, probes,
nucleotides, oligonucleotides, polynucleotides, etc.), droplets, encapsulated
cells,
encapsulated biomarkers, reagents, or any other suitable materials.
[0039] The system 100 can further additionally or alternatively include
any or all of
the system components as described in U.S. Application number 16/890,417 filed
02-JUN-
2020; U.S. Application number 16/867,235, filed 05-MAY-2020; U.S. Application
number
16/867,256, filed 05-MAY-2020; U.S. Application number 16/816,817, filed 12-
MAR-2020;
U.S. Application number 16/564,375 filed 09-SEP-2019; U.S. Application number
16/115,370, filed 28-AUG-2018; U.S. Application number 16/048,104, filed 27-
JUL-2018;
U.S. Application number 16/049,057, filed 30-JUL-2018; U.S. Application number
15/720,194, filed 29-SEP-2017; U.S. Application number 15/430,833, filed 13-
FEB-2017; U.S.
Application number 15/821,329, filed 22-N0V-2017; U.S. Application number
15/782,270,
filed 12-OCT-2017; U.S. Application number 16/049,240, filed 30-JUL-2018; and
U.S.
Application number 15/815,532, filed 16-NOV-2017, which are each incorporated
in their
entirety by this reference.
2.1 System ¨ Sample Cartridge and Sample Processing Chip
[0040] As shown in FIGURES iA and 2A-2C, the sample processing chip 132
functions
to provide one or more sample processing regions in which cells are captured
and optionally
sorted, processed, or otherwise treated for downstream applications, where the
downstream
applications can be performed at a sample processing cartridge 130 supporting
the sample
processing chip 132 (e.g., on-chip) and/or away from the sample processing
cartridge 130
(e.g., off-chip).
[0041] The sample processing cartridge 130 functions to support the
sample
processing chip 132, and to provide fluid pathways for fluid delivery,
capture, and sample
processing at the sample processing chip 132. The sample processing cartridge
130 can also
function to facilitate heat transfer to and from the sample processing chip
132 in relation to
sample processing procedures. Portions of the sample processing cartridge 130
can be
configured within a single substrate, but can additionally or alternatively
include multiple
portions (e.g. connected by fluidic pathways) across multiple substrates.
7
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0042] As shown in FIGURES 2A-2D, an example of the sample processing
cartridge
130' can include a base substrate 131 to which other elements are coupled
and/or in which
other elements are defined. Furthermore, in relation to sample processing
involving
microfluidic elements, the base substrate 131 can function as a manifold for
fluid transfer to
microfluidic elements, accessing of sample processing volumes at various
stages of
processing, and transfer of waste materials produced during sample processing.
In variations,
the base substrate 131 supports one or more of: the sample processing chip
132, an inlet
reservoir 133 for receiving sample material (e.g., containing cells,
containing particles, etc.)
and delivering it into the sample processing chip 132, an access region 134
for accessing one
or more regions of the sample processing chip 132, a lid 135 covering the
access region and
including a gasket 136 providing sealing functions, and a waste containment
region 137 for
receiving waste material from the sample processing chip 132. The cartridge
may have
additional gasketed ports to also connect with off-cartridge pumping systems.
Variations of
the base substrate 131 can, however, include other elements. For instance, as
described in
more detail below, the base substrate can include one or more openings,
recesses, and/or
protrusions that provide further coupling with the sample processing chip 132,
in order to
collectively define valve regions for opening and closing flow through the
sample processing
chip 132.
[0043] As shown in FIGURES 2A and 2C (bottom view), the sample processing
chip
132, (equivalently referred to herein as a microwell device or a slide)
defines a set of wells 103
(e.g. microwells) of a microwell region 34. Each of the set of wells can be
configured to capture
a single cell and/or one or more particles (e.g., probes, beads, etc.), any
suitable reagents,
multiple cells, or any other materials. In variations, microwells of the
sample processing chip
132 can be configured for co-capture of a single cell with a single functional
particle, in order
to enable analyses of single cells and/or materials from single cells without
contamination
across wells. Embodiments, variations, and examples of the sample processing
chip 132 are
described in one or more of: U.S. Application number 16/890,417 filed 02-JUN-
2020; U.S.
Application number 16/867,235, filed 05-MAY-2020; U.S. Application number
16/867,256,
filed 05-MAY-2020; U.S. Application number 16/816,817, filed 12-MAR-2020; U.S.
Application number 16/564,375 filed 09-SEP-2019; U.S. Application number
16/115,370,
filed 28-AUG-2018; U.S. Application number 16/048,104, filed 27-JUL-2018; U.S.
8
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
Application number 16/049,057, filed 30-JUL-2018; U.S. Application number
15/720,194,
filed 29-SEP-2017; U.S. Application number 15/430,833, filed 13-FEB-2017; U.S.
Application
number 15/821,329, filed 22-N0V-2017; U.S. Application number 15/782,270,
filed 12-OCT-
2017; U.S. Application number 16/049,240, filed 30-JUL-2018; and U.S.
Application number
15/815,532, filed 16-NOV-2017, which are each incorporated in their entirety
by reference
above.
[0044] In material composition, the sample processing chip 132 can be
composed of
microfabricated silicon or glass-fused silica materials, which function to
enable higher
resolution of the set of wells, enabled, for instance, by defining sharper
edges (e.g., thinner
well walls, well walls arranged at an angle approaching 90 degrees, etc.) in
the set of wells.
Material composition can further enable optical interrogation of contents of
the sample
processing chip 132 (e.g., through a bottom surface, through a top surface),
in relation to the
imaging subsystem 190 described in more detail below. Materials and
fabrication processes
described can further enable one or more smaller characteristic dimensions
(e.g., length,
width, overall footprint, etc.) of the microwell cartridge as compared to
conventional chip
designs. In specific examples, the sample processing chip 132 is fabricated
using deep reactive
ion etching (DRIE) techniques, according to specifications associated with one
or more of:
number of finished devices with acceptable level of defects (e.g., < 5%);
depth measured to
within +/- 1 micron of nominal depth (e.g., 25 microns); Rib measured to
within +/- 1 micron
of nominal rib dimensions (e.g., 5 microns). To mitigate any issues during the
fabrication,
specific examples of the sample processing chip 132 were developed with: a)
determination
of resist thickness and lithography required for etching glass substrates with
nominal depth
of 30 microns with nominal widths of 5 microns between microwells; b) lateral
resist erosion
and determination of mask bias; c) characterization of vertical taper of
microwell side-wall
after etching; and d) dicing process optimization to achieve good yield of
final devices.
[0045] Additionally or alternatively, the sample processing chip 132 can
include any
other suitable material, such as ¨ but not limited to ¨ a polymer, metal,
biological material,
or any other material or combination of materials. The sample processing chip
132 may be
fabricated by various processes such as precision injection molding, precision
embossing,
microlithographic etching, LIGA based etching, or by other suitable
techniques.
9
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0046] In some variations, one or more surfaces of the set of wells 103
(e.g., bottom
surface, side surface, bottom and side surfaces, all surfaces, etc.) can be
reacted with
oligonucleotide molecules for capture of biomarkers from individual cells into
individual
microwells. The oligonucleotide molecules present on each and individual
microwells may be
barcoded to allow biomarkers processed in each microwell to be linked back to
a particular
well and hence a particular single cell. In one variation, the set of wells
includes a set of
microwells having hexagonal cross sections taken transverse to longitudinal
axes of the wells,
as described in one or more of the applications incorporated by reference
above.
[0047] In one variation, as shown in FIGURE 2C, the sample processing
chip 132 can
include an inlet opening 32, a first fluid distribution network 33 downstream
of the inlet
opening, for distribution of fluids to a set of microwells 34 (e.g., 1,000 to
10,000,000 wells),
a second fluid distribution network 35 downstream of the set of microwells 34,
and an outlet
opening 36 coupled to a terminal portion of the second fluid distribution
network 35, for
transfer of waste fluids from the sample processing chip 132. In this
variation, the sample
processing chip 132 is coupled to a first side (e.g., under-side) of the base
substrate 131 (e.g.,
by laser welding, glue bonding, solvent bonding, ultrasonic welding or another
technique).
Coupling of the sample processing chip 132 to the side of the base substrate
131 can enable
transfer of heat from the heating and cooling subsystem 150 to the set of
microwells 34 and/or
other regions of the sample processing chip 132, where the heating and cooling
subsystem
150 is described in more detail below.
[0048] The base substrate 131, as described above, can also include an
inlet reservoir
133 (e.g., defined at a second side of the base substrate 131 opposing the
first side to which
the sample processing chip 132 is coupled). The inlet reservoir functions to
receive sample
material (e.g., samples containing cells, sample containing barcoded cells,
sample containing
encapsulated materials, samples containing particles, etc.) and/or sample
processing
materials from the process container 20' described above, for delivery into
the inlet opening
32 of the sample processing chip 132. In variations, the inlet reservoir 133
can be defined as
a recessed region within a surface of the base substrate 131, wherein the
recessed region
includes an aperture that aligns with and/or seals with the inlet opening 32
of the sample
processing chip 132. The inlet reservoir 133 of the base substrate 131 can
interface with
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
upstream fluid containing components and/or bubble mitigating components, as
described
in applications incorporated in their entirety by reference above.
[0049] In variations, one or more of the inlet reservoir 133 of the base
substrate 131
and the inlet 32 of the sample processing chip 132 can include valve
components that can be
open or closed by one or more components of the system 100. In a first
variation, the inlet
reservoir 132 includes an aperture that can be accessed by a pipette tip or
any other suitable
attachment of a fluid handling subsystem coupled to the gantry 170 (described
in more detail
below). In some embodiments, the aperture can be closed and therefore prevent
fluid from
traveling from the inlet reservoir 132 to the sample processing chip 132. The
inlet reservoir
132 can, however, be configured in another suitable manner. The opening
associated with the
inlet reservoir 133 may have a conical shape surface open towards the top
allowing interfacing
and sealing a pipette tip such that fluid (aqueous solutions or oil or air)
may be pumped
directly into the microchannel defined in 33 in FIGURE 2C.
[0050] As shown in FIGURES 2A and 2B, the base substrate 131 can also
define an
access region 134 for accessing one or more regions of the sample processing
chip 132, where
the access region can allow regions of the sample processing chip 132 to be
observed and/or
extracted from the sample processing chip 132 at various phases of sample
processing. As
shown in FIGURES 2A and 2B, the access region 134 can be defined as a recessed
region
within the base substrate 131, and include an opening 37 aligned with the
region of the sample
processing chip 132 that includes the set of microwells. The sample processing
chip 132 may
have as few as 100 microwells to as many as loo million microwells. As such,
in variations
wherein the microwell region is open to the environment (e.g., without a
covering to seal the
wells), the opening 37 of the access region 134 can function as a microwell to
provide access
to contents of the microwells for observation and/or material extraction
(e.g., by magnetic
separation, as described in further detail below). The opening 37 can match a
morphology
and footprint of the microwell region, and in a first variation, as shown in
FIGURE 2B, can
be a square opening. However, in other variations, the opening 37 can have
another suitable
morphology.
[0051] As shown in FIGURES 2A-2C, the base substrate 131 can include or
otherwise
couple to a lid 135 covering the access region 134, where the lid 135 can
include a gasket 136
providing sealing functions, and where the lid 135 functions to transition the
access region
11
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
134 between open and closed modes, thereby preventing evaporative sample loss
and/or
contamination of contents of the sample processing chip 132 during operation.
The lid 135
can additionally or alternatively function to protect the contents of the
microwells or other
processing regions of the sample processing chip 132 from debris, enable a
processing of the
contents of the sample processing chip 132 (e.g. by isolating regions from the
ambient
environment), initiate the start of a protocol (e.g., by opening to accept
reagents from a
pipettor), prevent user manipulation of the sample processing chip 132 (e.g.,
by closing after
all necessary reagents have been added), define (e.g., with the lid 135) part
or all of a fluid
pathway, cavity, or reservoir (e.g. serve as the top surface of a fluidic
pathway between the
inlet and the set of microwells, serve as a boundary of a fluid pathway
adjacent the microwell
region, serve as the top surface of a fluidic pathway between the set of wells
and the waste
chamber, etc.), or perform any other suitable function.
[0052] As shown in FIGURE 2B, in at least one variation, the lid 135 can
be
complementary in morphology to features of the access region 134, such that
the lid 135 mates
with the access region 134, while providing a gap with the sample processing
chip 132.
Additionally, in variations (shown in FIGURE 3B and 3C), the lid 135 can be
substantially
flush with the base substrate 131 at a top surface when the lid 135 is in the
closed position.
However, the lid 135 can be morphologically configured in another suitable
manner.
[0053] In variations, a protrusion 38 of the lid 135 can interface with
the opening 37
of the access region 134, thereby substantially preventing access to the
opening 37 when the
lid is in the closed position. As shown in FIGURE 2B, in some variations, the
protrusion 38
can have a base (or other region) surrounded by a gasket 136, which functions
to seal the
opening 37 of the access region 134 in the closed position of the lid 135.
Variations of the lid
135 can, however, omit a gasket and promote sealing of the access region 134
in another
suitable manner. In another embodiment, the entire bottom surface of the lid
that comes
closest to the microwells in the sample processing chip 132 can be an
elastomeric substrate
(e.g., flat elastomeric substrate) allowing the elastomeric lid to cover the
microwells, thereby
preventing any evaporative or diffusive loss of molecules during thermocycling
in each of the
microwells.
[0054] In some variations, the lid 135 can include a locking or latching
mechanism that
allows the lid 135 to be maintained in the closed position with the base
substrate 131 until the
12
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
locking/latching mechanism is released. In the variation shown in FIGURES 3A-
3C, a
peripheral portion of the lid 135 can include a one or more tabs 39 that
interface with
corresponding tab receiving portions of the base substrate 131, where, the
tabs 39 are
configured to flex when pushed into the base substrate 131 until they
interface with the tab
receiving portions of the base substrate 131 and return from a flexed
configuration to a latched
state. Additionally or alternatively, in the variation shown in FIGURES 3A-3C,
the
locking/latching mechanism can include a releasing body 41 (e.g., bar, recess,
hook, etc.) that
can be interfaced with in order to release the tab(s) 39 from the tab
receiving portions, and
transition the lid 135 from the closed mode to the open mode in relation to
the base substrate
131. As such, the lid 135 provides the lid an open mode in which the access
region 134 is
uncovered and a closed mode in which the access region 134 is covered. In the
variation shown
in FIGURES 3A-3C, the releasing element 41 includes a bar that is recessed
away from the
access region 134 of the base substrate 131, where the bar can be reversibly
coupled to a lid-
opening tool 145. In variations, the lid-opening tool 145 can include a first
region (e.g., first
end) that interfaces with a an actuator (e.g., actuating tip, pipettor of a
fluid handling
subsystem coupled to the gantry 170 described below, etc.), and a second
region (e.g., second
end) including a linking element 42 configured to interface with the releasing
element 41 of
the lid 135. Then, with movement of the pipettor/pipette interface, the lid-
opening tool 145
can be configured to pull on the releasing element 41 and/or push on the lid
135 in order to
transition the lid between open and/or closed modes. As such, in relation to
fluid handling
elements coupled to the gantry 170 described below, the system loo can provide
operation
modes for: coupling a lid-opening tool 145 to an actuator (e.g., coupled to a
gantry 170), the
lid-opening tool including a linking element 42; moving the lid-opening tool
into alignment
with a releasing element 41 of the lid 135, reversibly coupling the linking
element 42 with the
releasing element 41; and applying a force to the releasing element 41,
thereby releasing the
lid 135 from a latched state and transitioning the lid 135 from a closed mode
to an open mode.
In order to effectively apply an unlatching force (e.g., by the actuator
(e.g., coupled to a gantry
170), the base substrate 131 can be retained in position (e.g., by retention
elements described
in Section 2.1.4, by retention elements of the heating and cooling subsystems,
by retention
elements of the fluid level detection subsystem, by retention elements of the
deck, etc.) which
13
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
passively or actively apply counteracting forces against the unlatching forces
applied through
the lid-opening tool 145.
[0055] In variations, however, the locking/latching mechanism can
additionally or
alternatively include or operate by way of: a lock-and-key mechanism, magnetic
elements, or
another suitable mechanism. Furthermore, in alternative variations, the lid
135 can include
another lid actuator, for instance, including a motor that rotates the lid
about an access
parallel to a broad surface of the sample processing cartridge 130. The
actuator can
additionally or alternatively be configured to translate the lid 135 (e.g.
slide the lid 135 parallel
to a broad surface of the sample processing cartridge 130, translate the lid
135 perpendicular
to the broad surface, etc.) or otherwise move the lid 135 to selectively cover
and uncover one
or more predetermined regions (e.g. the set of microwells). As such, the lid
135 can be
configured to operate in an automated or semi-automated fashion, such that the
lid 135
automatically closes upon one or more triggers (e.g., cell capture protocol is
initiated by a
user, cell processing protocol is initiated by a user, all reagents for a
selected protocol have
been added from the process container 20, etc.) and opens upon one or more
triggers (e.g.,
cell capture protocol has been completed, upon user request, it has been
determined that the
cells are viable, it has been determined that single cells have been captured,
etc.). Additionally
or alternatively, operation of the lid 135 can be initiated and/or completed
by a user, operated
according to a schedule or other temporal pattern, or otherwise operated.
[0056] As shown in FIGURES 2A-2C, the base substrate 131 can also include
a waste
containment region 137 for receiving waste material from the sample processing
chip 132.
The waste containment region 137 can also function to maintain desired
pressures (e.g.,
vacuum pressures, etc.) within the sample processing chip 132, thereby
enabling flow of liquid
from the inlet reservoir 133 through the sample processing chip 132 and to the
waste
containment region 137. The waste containment region 137 can be defined as a
volume (e.g.,
recessed into the base substrate 131, extending from the base substrate 132,
coupled to an
outlet of the base substrate 131, etc.) for receiving waste or other materials
from the sample
processing chip 132. In the variation shown in FIGURES 2A-2C, the waste
containment
region 137 is defined at a side of the base substrate 131 opposing the side to
which the sample
processing chip 132 is coupled, such that waste from the sample processing
chip 132 is pushed
or pulled upward into the waste containment region 137 by forces of the
pumping subsystem
14
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
157 described in more detail below. However, the waste containment region 137
can
additionally or alternatively be configured in another suitable position
relative to the base
substrate 131 and the sample processing chip 132, in order to receive waste.
The waste
containment region 137 can have a volumetric capacity of 10-100 mL or another
suitable
volumetric capacity.
[0057] As shown in FIGURES 2A-2C, the waste containment region 137 can
include a
cover 48 (e.g., a cover that is approximately co-planar with the lid 135),
which facilitates
containment of waste within the waste containment region 137. Alternatively,
the waste
containment region 137 may not include a cover. Furthermore, as shown in
FIGURE 2C,
examples of the waste containment region 137 can include a pump outlet 51
distinct from the
cover, where the pump outlet 51 can allow the residual air in the waste
chamber to be
pressurized by an off-cartridge pump(e.g., by pumping mechanisms, etc.);
however,
variations of the waste containment region 137 can alternatively omit a waste
outlet.
[0058] In relation to the waste containment region 137, the system loo can
further
include a valve 43 configured to allow and/or prevent flow from the sample
processing chip
132 to the waste containment region 137. The valve 43 can interface with the
outlet opening
36 of the sample processing chip 132 described above, in order to enable
and/or Nock flow
out of the outlet opening 36 and into the waste containment region 137. The
valve 43 can have
a normally open state and transition to a closed state upon interacting with a
valve-actuating
mechanism. Alternatively, the valve 43 can have a normally closed state and
transition to an
open state upon interacting with a valve-actuating mechanism.
[0059] In the variation shown in FIGURES 2A and 4A-4B, the valve 43 can
include an
elastomeric body and is configured to couple the sample processing chip 132 to
the base
substrate 131 through an opening 44 of the sample processing chip 132 that
aligns with a
corresponding valve-receiving portion of the base substrate 131. In this
variation, a
transitionable portion of the valve 43 is configured to be positioned along a
flow path from
the outlet opening 36 of the sample processing chip 132 to the inlet of the
waste containment
region 137 of the base substrate 132 (e.g., along a flow path from the
microwell region to an
outlet of the sample processing chip into a waste containment region of the
sample processing
cartridge). In an example the opening 44 of the sample processing chip 132 is
contiguous with
the outlet opening 37 of the sample processing chip 132; however, in other
variations, the
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
outlet opening 37 and the opening 44 may be displaced from the each other and
connected by
another microfluidic channel. As such, closure of the valve 43 can Nock flow
from the outlet
opening 37 into the waste containment region 137, and the valve 43 can be
opened to allow
flow from the outlet opening 37 into the waste containment region 137.
[0060] In a variation shown in the cross sectional images of the base
substrate 131
shown in FIGURES 4A-4B, a valve actuator 45 can access the base substrate 131
from below
(e.g., from below the deck), and pass through a channel or other
recess/opening of the base
substrate 132 in order to interact with the valve 43. In particular, when a
tip 46 (aligned with
the opening into the base substrate) of the valve actuator 45 pushes against
the valve (e.g., a
elastomeric membrane of the valve 43), as shown in FIGURES 4B (top), the valve
43 can
transition to a closed state in order to fluidically decouple the outlet
opening 37 of the sample
processing chip 132 from the waste containment region 137. Additionally or
alternatively, as
shown in FIGURE 4B (bottom), removal of force by the valve actuator 45 can
remove pressure
from the valve 43 and transition it to an open state to fluidically couple the
outlet opening 36
of the sample processing chip 132 from the waste containment region 137. As
such, the valve
actuation subsystem includes an engaged mode wherein the tip extends into the
valve opening
to deform the elastomeric valve, thereby closing the flow path, and a
disengaged mode
wherein the tip is retracted, thereby opening the flow path. However, the
valve 43 can
additionally or alternatively be configured in another suitable manner.
[0061] In other variations, the system can include a similar mechanism
for coupling a
valve to other flow paths of the sample processing chip 132 and/or to the base
substrate 131.
[0062] Variations of the base substrate 131 can, however, include other
elements. For
instance, as described in more detail below, the base substrate 131 can
include one or more
openings, recesses, and/or protrusions that provide further coupling with the
sample
processing chip 132, in order to promote or inhibit flow through the sample
processing chip
132. For instance, as shown in FIGURE 4A, the base substrate 131 can include a
pump opening
46 that couples the base substrate 131 to a pumping element of the pumping
subsystem 157
(e.g., through deck no), in order to drive and/or stop fluid flow through the
sample
processing chip 132. The base substrate 131 of the sample processing cartridge
130 can,
however, include other suitable elements.
16
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0063] Embodiments, variations, and examples of the chip 101 can include
embodiments, variations, and examples of the capture devices described in one
or more
applications incorporated by reference above.
2.2. System ¨ Containers for Processing Retrieved Materials
[0064] In relation to processing (e.g., purification, washing,
extraction, amplification,
etc.) of retrieved material using the separation systems described, the system
loo can also
include a process container 20. The process container 20 functions to process
retrieved target
components of samples according to one or more workflows for various
applications, as
described in further detail below. As such, material can be retrieved from the
sample
processing chip 132 described above and transferred to the process container
20 for further
processing, as described in more detail below. Additionally or alternatively,
in some
variations, the process container 20 can contain, in one or more compartments,
materials for
cell capture and sample processing, in the context of a fully automated
system. As such, the
process container 20 can define a set of storage volumes distributed across a
set of domains,
where the set of domains can be configured for providing suitable environments
for the
material contents of each domain. The set of storage volumes can directly
contain sample
processing materials, and/or can alternatively be configured to receive and
maintain
positions of individual containers (e.g., tubes, etc.) that contain sample
processing materials.
The storage volumes of each domain can be distributed in arrays, or otherwise
arranged.
[0065] Individual storage volumes of the set of storage volumes of the
process
container 20 can further include one or more seals, which function to isolate
materials within
the process container 20, to prevent cross-contamination between materials
within
individual storage volumes, to prevent contaminants from entering individual
storage
volumes, and/or to prevent evaporative loss during storage and shipment. The
seal(s) can be
puncturable seal(s) (e.g., composed of paper, composed of a metal foil, and/or
composed of
any other suitable material). However, the seal(s) can alternatively be
configured to be non-
puncturable (e.g., the seal(s) can be configured to peel away from the process
container 20).
In embodiments, certain reagent containers may also be sealed by a hinged lid
that can be
opened or closed by a tool (e.g., as described in more detail below), as
needed for processing
at appropriate steps of the protocol.
17
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0066] In variations, the set of domains can include a first domain for
storing reagents
requiring a chilled environment (e.g., at a temperature from 1C-15C), a second
domain for
storing materials that can be stored in ambient conditions, a third domain
storing tubes with
materials for performing polymerase chain reaction (PCR) operations and
interfacing with
heating elements described below, a fourth domain for storing functionalized
particles (e.g.,
beads with probes having barcoding regions and other functional regions, as
described in U.S.
Application number 16/115,370, etc.), and a fifth domain for performing
separation
operations (e.g., separation of target from non-target material by magnetic
force). In
variations, domains providing different environments for the storage volumes
can be
configured differently. For instance, the first domain (i.e., for cold
storage) can be composed
of a thermally insulating material and/or can include insulating material
about storage
volumes of the domain (e.g., individually, about the entire domain).
Additionally or
alternatively, a domain for separation can be include magnetically conductive
materials
configured to provide proper magnetic field characteristics for separation.
Additionally or
alternatively, domains for thermocycling or other heat transfer applications
can be configured
with thermally conductive materials to promote efficient heat transfer to and
from the process
container 20. In embodiments, various domains can be optimally positioned such
that there
is minimal cross-talk between certain operations. For example, the domain(s)
for chilled
reagent storage volumes can be maintained a temperature (e.g., 4C) during a
run, whereas
the domain(s) for PCR reactions can require heating (e.g., up to 95C during
denature). As
such, to minimize the effect of PCR thermocycling on chilled reagents, the
domain(s)
containing the reagents stored at ambient temperature maybe configured in
between the PCR
thermocycling domain(s) and chilled domain(s). In order to further prevent
heat cross-talk,
additional buffer tubes with just air may be used in between critical domains
that need
independent temperature control.
[0067] In variations, process materials supported by the domains of the
process
container 20 can include one or more of: buffers (e.g. ethanol, priming
buffer, lysis buffer,
custom lysis buffers, sample wash buffers, saline with RNAase inhibitors, bead
wash buffers,
RT buffer, buffer, etc.), oils (e.g. perfluorinert oil), PCR master mixtures,
cells, beads (e.g.
functionalized beads) or any other suitable materials used for cell capture
and/or sample
processing. Additionally or alternatively, one or more of the set of storage
volumes can be
18
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
empty (e.g. initially empty, empty throughout one or more processes, empty
prior to filling
by an operator, etc.). Different storage regions in various domains of the
process container
20 can have initial reagent volumes from a few microliters (e.g., 5
microliters) to 50 milliliters.
[0068] In a specific example, as shown in FIGURE 5, the process container
20'
includes a first domain 21' at a first peripheral region of the process
container 20' for storing
reagents requiring a chilled environment, a second domain 22' at a central
region of the
process container 20' for storing materials that can be stored in ambient
conditions, a third
domain 23' at a peripheral region of the process container 20', near the
second domain 122,
for storing tubes with materials for performing polymerase chain reaction
(PCR) operations,
a fourth domain 24' at a peripheral region of the process container 20', for
storing
functionalized particles (e.g., beads with probes having barcoding regions and
other
functional regions, as described in U.S. Application number 16/115,370, etc.),
and a fifth
domain 25' at a peripheral region of the process container 20' for performing
separation
operations (e.g., separation of target from non-target material by magnetic
force). In the
specific example, the fourth domain 24' can be a modular element, whereby the
fourth
domain 24' can be stored separately from the rest of the process container 20'
until the
functionalized particles are ready for use, at which point the fourth domain
24' is set in
position and coupled with the process container 20'.
[0069] In the specific example, the first domain 21' and the second
domain 22' are
covered by a first seal composed of a metal foil, the third domain 23' and the
fifth domain 25'
are covered by a second seal composed of a paper, and the fourth domain 24' is
covered by a
third seal composed of a metal foil. However, variations of the example of the
process
container 20' can be configured in another suitable manner.
[0070] Furthermore, variations of the process container 20, 20' can omit
various
domains, and be configured for processing and separation of retrieved target
materials, as
described in more detail below.
[0071] The process container 20 can further additionally or alternatively
include
aspects described in applications incorporated by reference above.
2.3 System ¨ Deck, Separation Subsystem, and Gantry Aspects
[0072] In variations, aspects of the sample processing cartridge 130 and
process
container 20 can be supported by or otherwise interact with other system
elements (e.g., of a
19
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
system for automating sample processing). As shown in FIGURES 6A-6B and 7A-7C,
in
embodiments, the system 100 can include a deck 10, which functions as a
platform to support
and position one or more components of the system 100 (e.g., at a top broad
surface, at a top
and bottom broad surface, at a side surface, etc.) for automated sample
processing.
Furthermore, the deck 10 can function to position one or more components of
the system 100
to align with or otherwise interact with fluid processing subsystems, imaging
subsystems,
heating subsystems, separation subsystems (e.g., magnetic separation
subsystems), and/or
other subsystems coupled to the gantry 170 and/or base 180, as described
below. In this
regard, the deck 10 can be stationary as a reference platform, while other
components are
actuated into position for interacting with elements of the deck 10.
Alternatively, the deck 10
can be coupled to one or more actuators for positioning elements of the deck
10 for
interactions with other subsystems.
[0073] In the embodiment shown in FIGURES 6A-6B, the deck 110 provides a
platform supporting one or more units of the sample processing cartridge 130,
the process
container 20, a tool container 40 (described in applications incorporated by
reference), a
heating and cooling subsystem 50, a pumping subsystem 57, a fluid level
detection subsystem
59, a separation subsystem 160, and an imaging subsystem 90. The sample
processing
elements can be supported in a co-planar manner by the deck 10, or
alternatively at different
planes. Preferably, discrete elements supported by the deck are non-
overlapping, but
alternative embodiments of the deck no can support the sample processing
elements in an
overlapping manner (e.g., for conservation of space, etc., for operational
efficiency, etc.).
[0074] As such, and as shown in FIGURES 7A-7C, the deck 10 also includes
at least
one region for supporting a unit of the sample processing cartridge 130, where
the region
functions to position the sample processing cartridge 130 relative to portions
of the heating
and cooling subsystem 50, the pumping subsystem 57, the fluid level detection
subsystem 59,
and/or the imaging subsystem 90. In this regard, the region can include one or
more
openings, recesses, and/or protrusions for providing interfaces between
complementary
portions of the sample processing cartridge 130 and associated portions of the
heating and
cooling subsystem 50, the pumping subsystem 57, the fluid level detection
subsystem 59, and
the imaging subsystem 90, and additionally to promote and maintain alignment
between
such portions.
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0075] Similarly, as shown in FIGURES 7A-7C, the deck 10 can include at
least one
region for supporting a unit of the process container 20, where the region
functions to
position the process container 20 relative to portions of the heating and
cooling subsystem
50, and separation subsystem 160 described in more detail below. In this
regard, the region
can include one or more openings, recesses, and/or protrusions for providing
interfaces
between complementary portions of the process container and associated
portions of the
heating and cooling subsystem 50 and separation subsystem 60, and additionally
to promote
and maintain alignment between such portions.
[0076] As shown in FIGURES 7A and 7B, the deck 10 can also include at
least one
region for supporting a unit of a tool container 40, where the region
functions to position the
tool container 40 relative to fluid handling apparatus of the gantry 170
described below.
Embodiments, variations, and examples of the tool container 40 are described
in applications
incorporated by reference above.
[0077] Embodiments, variations, and examples of the heating and cooling
subsystem
50, pumping subsystem 57, fluid level detection subsystem 59, and imaging
subsystem 90,
and coupling with regard to gantry 170 (with pipettor 174) and base 180 are
also described in
more detail in applications incorporated by reference. Aspects of the
separation subsystem
160 are further described below.
2.4 System ¨ First Variation for Retrieval by Magnetic Force
[0078] As shown in FIGURES 8A-8C, a variation of the system 200 includes
an
adaptor 210 (e.g., of separation subsystem 160 described above) including a
first region 211
configured to couple to a capture region of a sample processing cartridge
(e.g., embodiment
of sample processing cartridge 130 described above), for capturing particles
in single-particle
format, a second region 212, and an internal cavity 220 passing from the first
region to the
second region; a magnet 230 configured to pass into the internal cavity 220 of
the adaptor
210 and apply an attracting force to the capture region of the sample
processing cartridge 130
during operation; and a support structure 240 reversibly coupled to the second
region 212 of
the adaptor 210 and to the magnet 230.
[0079] The support structure 240 of the system 200 can also include a
plunger
subsystem 250 coupled to an ejector proximal the magnet 230, wherein in a
baseline
operation mode the adaptor 210 is coupled to the support structure 240 and the
plunger
21
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
subsystem 250 is not activated, and in an ejecting mode, the adaptor 210 is
released from the
support structure 240 in response to activation of the plunger subsystem 250.
In variations,
the plunger subsystem 250 can also include structures that function to
facilitate fluid
dispensing and aspiration functions in order to dispense and/or retrieve
material from the
capture region of the sample processing cartridge 130. Furthermore, the system
200 can
include a guide 260 configured to retain the support structure 240 in position
relative to the
capture region of the sample processing cartridge i3oand to prevent physical
contact between
the magnet 230 and the capture region of the sample processing cartridge
i3oduring
operation.
[0080] The system 200 functions to controllably apply a magnetic force to
the capture
region of the chip 201, in order to provide an attractive force for drawing
target material
coupled (directly or indirectly) to magnetic components within the capture
region, into the
adaptor 210. Embodiments of methods implemented with the system 200 can
produce
retrieval of target material in 5-8 minutes of manual operation time (and 10-
45 minutes total
time), with a retrieval efficiency of >90% where only magnetic particles
coupled to target
material of the sample are retrieved. The system 200 can thus function to
produce increased
selective retrieval efficiency can thus reduce downstream costs in relation to
processing
reagent and other material costs (due to reduced volumes needed, due to
reduced splits in
biochemistry reactions) and processing burden. The system 200 can implement
one or more
embodiments, variations, or examples of the method(s) described below, and/or
can be used
to implement other methods.
2.4.1 First Magnetic Variation - Adaptor
[0081] As shown in FIGURES 8A-8C, the adaptor 210 includes a first region
211
configured to couple to a capture region of a sample processing chip 132 for
capturing
particles in single-particle format, a second region 212, and an internal
cavity 220 passing
from the first region to the second region. The adaptor 210 functions to
provide structures
that separate the magnet 230 from physically contacting wells or other
sensitive material at
the capture region of the sample processing cartridge 130, and to support
application of a
magnetic field to the capture region for retrieval of target material of the
sample processing
cartridge 130 by the system 200, by transmitting magnetic forces to a region
of the adaptor
210 interfacing with the capture region of the sample processing cartridge
130. The adaptor
22
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
210 can also function to prevent sample cross contamination, by serving as a
disposable
component that can be discarded between uses of the system 200. Units of the
adaptor can
be stored at an embodiment of the tool container 40 supported by the deck 10
described
above, or can be stored or staged by the system in another suitable manner. As
shown in
FIGURE 8A, units of the adaptor 210 can be retained in position within a rack
or portion of
the tool container 40 until they are needed for use.
[0082] The adaptor 210 can be morphologically prismatic with an internal
cavity 220,
where the cross section of the adaptor 210 along its longitudinal axis is
defined by a polygonal
perimeter, an ellipsoidal perimeter, an amorphous perimeter, or a boundary of
any other
suitable shape (e.g., closed shape, open shape). The cross section of the
adaptor 210 can
complement a shape of a footprint of the capture region of the sample
processing cartridge
130, but may alternatively not complement a shape corresponding to the capture
region of
the sample processing cartridge 130. The adaptor 210 can have a length from
0.5-8 cm and a
width from 0.2-4 cm (e.g., corresponding to the shape of the capture region of
the chip 201).
The adaptor 210 preferably has a wall thickness that supports application of a
magnetic force,
from the magnet 230, to the capture region interfacing with the first region
211 of the adaptor
210. The wall thickness can be constant or non-constant along the length of
the adaptor 210.
In examples, the wall thickness can range from 0.2 to 3 mm thick; however, in
other examples,
the wall thickness can have any other suitable thickness. The surface of the
adaptor that
receives the magnetic particles is made smooth (e.g., surface finish better
than SPIBi) such
that the small magnetic particles (1-3 micron) do not gets entrapped in the
surface during the
bead capture onto its surface and subsequent release to another receptacle.
[0083] The adaptor 210 can additionally or alternatively include
structural features
that enable operation modes of the system 200. For instance, in relation to
release of the
adaptor 210 from the support structure 240 (described in more detail below),
the adaptor 210
can include a protrusion 214 configured to interface with the plunger
subsystem 250, where
a trigger of the plunger subsystem 250 can push against the protrusion 214 to
release the
adaptor 210 from the support system 240, once the plunger subsystem 250 is
activated. The
protrusion 214 can be a rim about the second region 212 of the adaptor 210, or
can
alternatively be defined by any other suitable morphology.
23
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0084] As described above, the adaptor 210 interfaces, at a first region
211, with an
exposed capture region of the sample processing cartridge 130 (e.g., with lid
135 open to
provide access to access region 134), in order to facilitate application of
magnetic force to the
capture region, and to enable drawing of target material (e.g., target
material coupled to
magnetic particles) into the adaptor 210 for further downstream processing.
The adaptor 210
can thus include a seal at the first region 211, in order to facilitate
mechanisms for drawing
target material from the sample processing chip 132 to the adaptor 210. The
seal can be a
separate element or an element integrated with the adaptor 210. The adaptor
210 can,
however, omit a seal at the first region 211. The adaptor 210 also couples, at
a second region
212, to the support structure 240, for retention in position relative to the
magnet 230, and for
reversible coupling and removal from the support structure 240. Coupling of
the adaptor 210
to other system components can occur with one or more of: a press fit, a snap
fit, a friction fit,
a male-female coupling interface, a screw, another fastener, a magnetic
mechanism, and any
other suitable mechanism.
[0085] The adaptor 210 can be composed of a polymeric material (e.g.,
plastic) that
does not adversely affect the magnetic field applied by the magnet 230 during
operation. The
adaptor 210 can additionally or alternatively include (e.g., include particles
of) or be
composed of a material (e.g., metallic material) that is magnetic or can
produce an induced
magnetic field to support applications of use of the system 200. The adaptor
210 can
additionally or alternatively be composed of any other suitable material.
Distributions of the
material(s) of the adaptor 210 can be homogenous or non-homogenous through the
body of
the adaptor, in relation to desired magnetic effects at the capture region of
the chip 201. The
internal cavity 220 of the adaptor 210 can include a medium (e.g., magnetic
medium, etc.), or
can alternatively not include any medium.
2.4.2 First Magnetic Variation - Magnet
[0086] As shown in FIGURES 8A-8C, the magnet 230 is configured to pass
into the
internal cavity 220 of the adaptor 210 and apply an attracting force to the
capture region and
target material captured at the sample processing cartridge 130 during
operation. The magnet
230 functions to generate a magnetic field that can attract target material
captured at the
capture region of the sample processing chip 132 (e.g., coupled to magnetic
particles within
the capture region) toward the adaptor 210 for further processing. The magnet
shape and pole
24
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
configuration is such that nearly normal magnetic force is applied to majority
of the target
microwells from where entrapped particles are being removed.
[0087] The magnet 230 can be morphologically prismatic, where the cross
section of
the magnet 230 along its longitudinal axis is defined by a polygonal
perimeter, an ellipsoidal
perimeter, an amorphous perimeter, or a boundary of any other suitable shape
(e.g., closed
shape, open shape). In variations, the magnet can have a length from 0.25-5"
and a width
from 0.1-1". In a specific example, the magnet has a square cross section
along its length and
has a length of 2" and sides of 0.25" width. The magnet 230 of the specific
example has a
weight of 15.4g.
[0088] The magnet 230 couples, at a first end, to the support structure
240, and passes
into the internal cavity 210 of the adaptor 210. In variations, as shown in
FIGURE 2B, units
of the magnet 230 (and corresponding adaptor 210) can have different
dimensions to support
different variations of the chip 201. For instance, a unit of the magnet 230
can have a small
cross section (e.g., 0.25"x0.25") to support a chip variation with fewer
microwells, and a unit
of the magnet 230 can have a larger cross section (e.g., 0.375"x0.375" to
support a chip
variation with more microwells).
[0089] The magnet 230 is composed of a permanent magnetic material, but
can
alternatively be an electromagnet. In variations, the magnet 230 can be
composed of one or
more of: alnico, neodymium, neodymium iron boron, samarium cobalt, ferrite,
and any other
suitable magnetic material. The magnet 230 can additionally or alternatively
include a plating
material, in order to facilitate operations involving processing of biological
samples or other
samples. In a specific example, the magnet 230 is composed of neodymium iron
boron
(NdFeB, grade 42) with a nickel-based coating (e.g., nickel-copper-nickel
coating).
[0090] The magnet 230 can have one or more magnetization directions, and
in
variations, can produce a pull and/or push force up to 10 lbs., with a surface
field of up to
12,000 Gauss, an internal field up to 30,000 Gauss (e.g., BRmax of 30,000
Gauss), and an
energy density (BHmax) of up to 90 MG0e. In a specific example, the magnet 230
has a
magnetization direction through its thickness, a pull force of 5.58 lbs., a
surface field of 6584
Gauss, a BRmax of 13,200 Gauss, and a BHmax of 42 MG0e. In terms of field, the
magnet
230 of the specific example is magnetized through its length so the poles are
one the
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
0.25"X0.25" ends of the magnet 230. However, the magnet 230 can alternatively
be
configured to produce any other suitable field.
2.4.3 First Magnetic Variation - Support Structure
[0091] As shown in FIGURES 8A and 8C, the support structure 240 is
reversibly
coupled to the second region of the adaptor 210 and to the magnet 230. The
support structure
functions to retain the magnet 230 in position, and to transition between
operation modes
for coupling and uncoupling the magnet 230 and adaptor 210. The support
structure 240 can
also function to transition between operation modes for drawing material out
of the capture
region of the sample processing cartridge 130 for downstream processing.
[0092] The support structure 240 can have a housing with a form factor
similar to that
of a manual pipettor, where the housing has a surface with a gripping region
(e.g., series of
protrusions and recesses) configured to complement a user's hand.
Alternatively the support
structure 240 can be configured to not be handled by a human operator, and can
additionally
or alternatively include features for interfacing with a robotic apparatus
(e.g., interface of
pipettor 174 coupled to gantry 170 described above) for automated target
material retrieval
from the capture region of the sample processing cartridge 130. A variation of
this
embodiment is described in more detail below, with various operation modes for
material
retrieval and processing.
[0093] As noted above, the support structure 240 of the system 200 can
also include a
plunger subsystem 250 coupled to an ejector proximal the magnet 230, wherein
in a baseline
operation mode the adaptor 210 is coupled to the support structure 240 and the
plunger
subsystem 250 is not activated, and in an ejecting mode, the adaptor 210 is
released from the
support structure 240 in response to activation of the plunger subsystem 250.
As described
above, the ejector can interface with the protrusion 214 of the adaptor 210,
in order to release
the adaptor 210 from the support structure 240 in the ejecting mode.
Furthermore, in
variations, the plunger subsystem 250 can also include structures that
function to facilitate
fluid dispensing and aspiration functions in order to dispense and/or retrieve
material from
the capture region of the chip 201. As such, variations of the plunger
subsystem 250 can
perform similar functions to that of a pipettor, in addition to supporting
magnetic field
application and adaptor release.
26
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[0094] The support structure 240 can be composed of one or more polymeric
materials
(e.g., plastics) that are sanitizable (e.g., autoclavable, resistant to damage
by ethanol, etc.)
between uses of the system 200. However, the support structure 240 can
alternatively be
composed of another suitable material.
2.4.4 First Magnetic Variation - Guide
[0095] As shown in FIGURES 8A and 8C, the system 200 can include a guide
260
configured to retain the support structure 240 in position relative to the
capture region of the
sample processing chip 132 and to prevent physical contact between the magnet
230 and the
capture region of the sample processing chip 132 during operation. The guide
260 thus
functions to provide support about the support structure 240 and/or chip 201,
during the
retrieval process. As such, in one variation, the guide 260 can include
recessed regions
configured to receive the support structure 240 with coupled magnet 230 and
adaptor 210,
as well as the chip 201, in order to fix relative alignment between the
capture region of the
sample processing chip 132 and the adaptor 210. The guide ensures that the
adaptor surface
is placed above the microwells of the sample processing cartridge 130 by a
fixed distance,
(e.g., 25 microns, 100 microns, 0.5 rrirrl, or 1 rrirrl, or 2 rrirrl, or 3
rrirrl, or 4 rrirrl, 5 rrirrl, 6 rrirrl,
etc.). In another variation, as shown in FIGURE 8C, the guide 260 can be
configured to couple
to the sample processing cartridge 130 and to position the adaptor 210
relative to the capture
region of the chip 201, by only contacting the adaptor. The guide 260 can,
however, be coupled
to any other suitable portion of the system 200 to provide support.
[0096] The guide 260 can be composed of one or more polymeric materials
(e.g.,
plastics) that are sanitizable (e.g., autoclavable, resistant to damage by
ethanol, etc.) between
uses of the system 200. However, the guide 260 can alternatively be composed
of another
suitable material. The guide 260 can also be a disposable portion of the
system 200.
2.5 System ¨ Second Variation for Retrieval and Processing by Magnetic
Force
[0097] As shown in FIGURES 6A, 7B, and 9A-9C, a variation of the system
200' can
include a variation of adaptor 210', magnet 230', and support structure 240'
(e.g., of
separation subsystem 160, which functions to facilitate separation and
retrieval of target
material from non-target material using magnetic forces. In variations, the
separation
subsystem 160 can include embodiments, variations, and examples of components
described
27
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
in applications incorporated by reference above and described in further
detail below.
However, other variations of the separation subsystem 160 can additionally or
alternatively
include other components.
2.5.1 Second Magnetic Variation - Adaptor
[0098] As shown in FIGURES 9A-9C, the adaptor 210' can include a first
region 211
configured to interface with the sample processing chip 132, for instance,
through access
region 134, in order to enable retrieval of target material from the sample
processing chip 132.
The adaptor 210' can also include a second region 212' for coupling with the
magnet 230' (e.g.,
magnetic distal portion) of support structure 240', and an internal cavity
220' passing from
the first region to the second region. The adaptor 210' functions to provide
structures that
separate the magnet 230' and/or support structure 240' from physically
contacting wells or
other sensitive material of the sample processing chip 132, and to support
application of a
magnetic field to the desired regions for retrieval of target material (or non-
target material).
The adaptor 210' can also function to prevent sample cross contamination, by
serving as a
disposable component that can be discarded between uses of the system 200'.
[0099] The adaptor 210' can be morphologically prismatic with an internal
cavity 220',
where the cross section of the adaptor 210' along its longitudinal axis is
defined by a polygonal
perimeter, an ellipsoidal perimeter, an amorphous perimeter, or a boundary of
any other
suitable shape (e.g., closed shape, open shape). The cross section of the
adaptor 210' can
complement a shape of a footprint of the microwell region of the sample
processing chip 132,
but may alternatively not complement a shape corresponding to the sample
processing chip
132. The adaptor 210' preferably has a wall thickness that supports
application of a magnetic
force, from the magnet 230', to the sample processing chip 132 interfacing
with the first
region 211' of the adaptor 210'. The wall thickness can be constant or non-
constant along the
length of the adaptor 210. In examples, the wall thickness can range from 0.2
to 3 mm thick;
however, in other examples, the wall thickness can have any other suitable
thickness. The
surface of the adaptor 210' that receives the magnetic particles is made
smooth (e.g., surface
finish better than SPIBi) such that small functionalized particles (e.g., 1-3
micron in
characteristic dimension) do not get entrapped at the surface during capture
and subsequent
release to another receptacle (e.g., process container 20).
28
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00100] The adaptor 210' can additionally or alternatively include
structural features
that enable separation operation modes of the separation subsystem 160,
described in more
detail below. For instance, in relation to release of the adaptor 210' from
the support structure
240', the adaptor 210' can include a protrusion 214' configured to allow
another object (e.g.,
sleeve stripping tool 165 described in more detail below) to provide a force
against the
protrusion 214' to release the adaptor 210' from the support structure 240'.
[00101] As described above, the adaptor 210' interfaces, at a first region
211', with a
capture region of the sample processing chip 132 exposed through access region
134, in order
to facilitate application of magnetic force to the region, and to enable
drawing of material
(e.g., target or non-target material coupled to magnetic particles) to the
adaptor 210' for
further downstream processing. The magnetic sleeve 1410 can thus include a
seal at the first
region 211', in order to facilitate mechanisms for drawing target material
from the sample
processing chip 132 to the adaptor 210'. The seal can be a separate element or
an element
integrated with the adaptor 210'. The adaptor 210' can, however, omit a seal
at the first region
211'.
[00102] The adaptor 210' can be composed of a polymeric material (e.g.,
plastic) that
does not adversely affect the magnetic field applied by the magnet 230' during
operation. The
adaptor 210' can additionally or alternatively include (e.g., include
particles of) or be
composed of a material (e.g., metallic material) that is magnetic or can
produce an induced
magnetic field to support applications of use of the system 200'. The adaptor
210' can
additionally or alternatively be composed of any other suitable material.
Distributions of the
material(s) of the adaptor 210' can be homogenous or non-homogenous through
the body of
the adaptor, in relation to desired magnetic effects at the capture region of
the sample
processing chip 132. The internal cavity 220' of the adaptor 210' can include
a medium (e.g.,
magnetic medium, etc.), or can alternatively not include any medium.
2.5.2 Second Magnetic Variation ¨ Support Structure and Magnet
[00103] In the variation shown in FIGURES 9A-9C, the system 200' can
include a
support structure 240' including an interface 162 to the fluid handling
subsystem (e.g., pipette
interface) of the gantry 170 described above, and magnet 230' configured to
provide magnetic
forces for target material separation. In this variation, the magnet 230' can
be configured to
couple with one or more units of the adaptor 210', in variations where the
adaptor 210' is a
29
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
disposable elements. Furthermore, the interface 162 can be configured to
couple to a pipetting
head coupled to the gantry 170 described in more detail below, in order to
facilitate
automation of target or non-target material retrieval by way of magnetic
forces, fluid
aspiration, and/or fluid delivery operations provided by the pipetting head.
As such, the
system loo can include a separation mode in which the gantry 170 transports
the support
structure 240', coupled to the adaptor 210', between units of the sample
processing cartridge
130 and the process container 20 for magnetic separation and processing of
target material
from a sample. Furthermore, embodiments of methods implemented using the
separation
subsystem 160 can produce rapid retrieval of target material, with a retrieval
efficiency of
>90% where only magnetic particles coupled to target material (or non-target
material) of the
sample are retrieved. The separation subsystem 160 can thus function to
produce increased
selective retrieval efficiency can thus reduce downstream costs in relation to
processing
reagent and other material costs (due to reduced volumes needed, due to
reduced splits in
biochemistry reactions) and processing burden.
[00104] As shown in FIGURE 9A, the support structure can include an
interface 162 to
the fluid handling subsystem of the gantry 170 described below, where the
interface includes
a coupling region that complements a corresponding coupling region of the
fluid handling
subsystem. The coupling region of the interface 162 can operate by: a magnetic
coupling
mechanism; a press fit; a snap fit, a screwing mechanism; a male-female
connection; or
another suitable mechanism for providing reversible coupling with the fluid
handling
subsystem.
[00105] The magnet 230' of the support structure 240' can include or be
composed of a
material for providing a permanent magnet, or can alternatively be configured
as an
electromagnet (e.g., with coupling to suitable electronics of the system loo).
In variations, the
magnetic distal region 163 can be composed of one or more of: alnico,
neodymium,
neodymium iron boron, samarium cobalt, ferrite, and any other suitable
magnetic material.
In morphology, the magnet 230' can complement a morphology of the adaptor
210', such that
units of the adaptor 210'can couple (e.g., reversibly couple) with the magnet
230'.
Furthermore, the morphology and pole configuration of the magnet 230' is such
that nearly
normal magnetic force is applied to majority of the target microwells from
where entrapped
particles are being removed.
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
2.5.3 Second Magnetic Variation ¨ Optional Separation Elements Involving
Deck,
Gantry, and/or Base
[00106] As shown in FIGURES 6A, 7B, and loA-10B, in variations, the
separation
subsystem 160 can include a magnet subsystem 166 including a set of magnets
167 within a
housing 168, where the magnet subsystem 166 further includes a magnet actuator
169
configured to move the set of magnets 167 relative to the deck 10 (e.g.,
through in opening in
the deck Do), and into/out of alignment with one or more separation reservoirs
129a, 129 of
the process container 20 described above. The magnet actuator 169 can also be
coupled to
control circuitry (e.g., at the base 180). Furthermore, the magnet actuator
169 can be
configured to transition the set of magnets between a retracted state and an
extended state,
wherein in the extended state, the set of magnets passes into the first region
of the deck (e.g.,
as shown in FIGURES loA and 10B). As such, the separation subsystem 160 can
also include
elements that are supported by the deck 10 and/or base 180, in order to enable
operations for
separating target material from non-target material.
[00107] In variations, the set of magnets 167 can include one or more
permanent
magnets and/or electromagnets (e.g., with coupling to suitable electronics of
the system Dm).
Permanent magnets can be composed of one or more of: alnico, neodymium,
neodymium iron
boron, samarium cobalt, ferrite, and any other suitable magnetic material.
[00108] In the example shown in FIGURES 10A-10B, the set of magnets 167
can include
a first subset of magnets 167a arranged in a linear array (e.g., for
performance of purification
operations at the process container 20% where the positions of the first
subset of magnets
167a correspond to positions of volumes of the fifth domain 25' for particle
separation/purification, described in relation to the process container 20'
above and
workflows described in Section 3 below. In the example shown in FIGURE 10A-
10B, the set
of magnets 167 also includes a second subset of magnets 167b (e.g., one or
more magnets)
displaced from or otherwise offset from an axis associated with the first
subset of magnets
167a, in order to interact with a separation reservoir 129, 129a of the
process container 20
(e.g., for initial bead retrieval). The set of magnets 167 can, however, be
arranged in another
suitable manner (e.g., in relation to distributed arrays, in relation to
number, etc.) in relation
to providing suitable interactions with separation reservoirs 129 of the
process container 20
or other containers.
31
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00109] The housing 168 functions to surround the set of magnets 167, and
to provide
smooth operation in relation to transitioning the set of magnets 167 into/out
of alignment
with corresponding portions of the process container 20, 20'. Thus, as shown
in FIGURE loB,
in relation to configurations where there is a first subset of magnets 167a
and a second subset
of magnets 167b, the housing 168 can include a first surface (e.g., first
planar surface) tracking
the first subset of magnets 167a, and a second surface (e.g., second planar
surface) tracking
the second subset of magnets 167b, wherein the first surface 168a and the
second surface 168b
are angled away from each other. In this variation, a pair of opposing walls
can extend from
the first surface and the second surface, in order to promote smooth operation
(e.g., sliding
operations) of the housing 168 and magnets through the deck 10 in order to
interface with the
process container 20, 20'.
[00110] In relation to the process container 20', as shown in FIGURE loB,
volumes of
the process container 20' configured for magnetic separation can each include
a planar
surface 128a, or other surface complementary to the housing 168 at sides
configured to be
closest to the housing 168 during operation (e.g., in the extended magnet
states).
Furthermore, volumes of the process container 20' configured for magnetic
separation can
each include a second surface 128b (e.g., curved surfaces) displaced away from
the housing
168 for aspiration and/or delivery of fluids by a pipettor coupled to the
gantry 170. Cross
sections taken longitudinally through separation volumes/reservoirs 129, 129a
of the reagent
cartridge 120 can further be tapered toward a base of the process container
20, 20', such that
separation operations require a lower volume of fluid and/or provide more
efficient
aspiration and separation of target from non-target material.
2.5.4 Second Magnetic Variation ¨ Operation Modes
[00111] As shown in FIGURES nA through 11J, the separation subsystem 160
can
provide a sequence of operation modes for material separation, where, as shown
in FIGURE
12A, the operation modes involved specific system structure configurations of:
a support
structure 240' coupled with a pipetting head or other actuatable component
(e.g., interface of
pipettor 174 coupled to gantry 170), the support structure 240' coupled to a
magnet 230', a
unit of adaptor 210', a sleeve stripping tool 165, a separation reservoir 129,
and a magnet 167b
of the set of magnets 167 described above.
32
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00112] In more detail, as shown in FIGURE iiB, the separation subsystem
160 can
provide a first operation mode 164a, where the first operation mode 164a is a
baseline
operation mode in which the support structure 240' is uncoupled from a pipette
interface or
other actuatable component (e.g., described in relation to the gantry 170
below) and the
magnet 230' of the support structure 240' is uncoupled from the adaptor 210'.
The magnetic
sleeve 1410 is further retained by sleeve stripping tool 165 above the
separation reservoir 129
(or in variations, at another position), and the magnet 167b is displaced away
from the
separation reservoir 129 (e.g., by magnet actuator 169 described above). As
such, in the first
operation mode 164a, the system can stage the adaptor 210' in position near
the separation
reservoir 129, and stage the support structure 240' coupled to magnet 230' in
preparation for
separation and retrieval of target material from a sample.
[00113] As shown in FIGURE liC, the separation subsystem 160 can provide a
second
operation mode 164b, where the second operation mode 164b is an initializing
operation
mode in which the support structure 240' is coupled with a pipette interface
or other actuator
interface 6 (e.g., described in relation to the gantry 170 and pipettor 174)
and the magnet 230'
of the first body 161 is uncoupled from the adaptor 210'. The adaptor 210' is
further retained
by sleeve stripping tool 165 above the separation reservoir 129, and the
magnet 167b is
displaced away from the separation reservoir 129 (e.g., by magnet actuator 169
described
above). As such, in the second operation mode 164b, the system stages the
adaptor 210' in
position near the separation reservoir 129, and couples the support structure
240', with
magnet 230', to the pipettor 174 (e.g., through actuator interface 6) in
preparation for
separation and retrieval of target material from a sample.
[00114] As shown in FIGURE iffi, the separation subsystem 160 can provide
a third
operation mode 164c, wherein, in the third operation mode 164c, the support
structure 240'
is coupled with a pipette interface or other actuator interface 6 (e.g.,
described in relation to
the gantry 170 and pipettor 174) and moved into alignment with the separation
reservoir 129.
In the third operation mode 164c, the magnet 230' of the support structure
240' is coupled
with the adaptor 210' above the separation reservoir 129 in the retained
position of the
adaptor 210'. In the third operation mode 164c, magnet 167b is displaced away
from the
separation reservoir 129 (e.g., by magnet actuator 169 described above). As
such, in the third
operation mode 164c, the system transitions the support structure 240' and
magnet 230' (e.g.,
33
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
by way of pipettor 174) for coupling with the adaptor 210' retained near the
separation
reservoir 129, in preparation for separation and retrieval of target material
from a sample.
[00115] As shown in FIGURE nE, the separation subsystem 160 can provide a
fourth
operation mode 164d, wherein, in the fourth operation mode 164d, the support
structure 240'
is coupled with a pipette interface or actuator interface 6 (e.g., described
in relation to the
gantry 170 and pipettor 174) and the magnet 230' of the support structure 240'
is coupled
with the adaptor 210' above the separation reservoir 129. In the fourth
operation mode 164d,
the pipetting head (or other actuatable component) moves the support structure
240' and
magnet 230' coupled to the adaptor 210' out of the retained position provided
by the sleeve
stripping tool 165, to prepare for attraction of material (e.g.,
functionalized particles from the
sample processing cartridge) derived from the sample. In the fourth operation
mode 164d,
magnet 167b is displaced away from the separation reservoir 129 (e.g., by
magnet actuator
169 described above). As such, in the fourth operation mode 164d, the system
transitions the
support structure 240' and magnet 230' (e.g., by way of pipettor 174), coupled
to the adaptor
210', out of the retained position and into separation reservoir 129, in
preparation for
separation and retrieval of target material from a sample.
[00116] As shown in FIGURE 11F, the separation subsystem 160 can provide a
fifth
operation mode 164e, wherein, in the fifth operation mode 164e, the support
structure 240'
is coupled with a pipette interface or actuator interface 6 (e.g., described
in relation to the
gantry 170 and pipettor 174) and the magnet 230' is coupled to the adaptor
210' within the
separation reservoir 129. In the fifth operation mode 164d, the pipette
interface (or other
actuatable component) delivers fluid derived from the sample (e.g., lysed
target material
bound to target particles) into the separation reservoir 129, and the adaptor
210', still coupled
with the support structure 240', is submerged within the fluid in the
separation reservoir 129
to attract functionalized particles bound to target content. In the fifth
operation mode 164e,
magnet 167b is displaced away from the separation reservoir 129 (e.g., by
magnet actuator
169 described above). As such, in the fifth operation mode 164e, the system
configures the
support structure 240', magnet 230', and adaptor 210', for attraction of
target material
delivered into the separation reservoir 129.
[00117] As shown in FIGURE 11G, the separation subsystem 160 can provide a
sixth
operation mode 164f, wherein, in the sixth operation mode 164f, the support
structure 240'
34
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
is coupled with a pipette interface or actuator interface 6 (e.g., described
in relation to the
gantry 170 and pipettor 174) and the magnet 230' of the first body 161, still
coupled with the
adaptor 210', is moved by back into a retained position at the sleeve
stripping tool 165. In the
sixth operation mode 164f, the adaptor 210' (still coupled with target
material/functionalized
particles) is submerged within the fluid in the separation reservoir 129. In
the sixth operation
mode 164f, magnet 167b is displaced away from the separation reservoir 129
(e.g., by magnet
actuator 169 described above). As such, in the sixth operation mode 164f, the
system
configures the support structure 240', magnet 230', and adaptor 210', for
processing of target
material bound to the adaptor 210'. For instance, while material is bound to
the adaptor, the
system can perform wash steps to remove non-target material, or other
processes.
Additionally or alternatively, the sixth operation mode 164f can prepare
target material for
transfer from the adaptor 210' to a region of the separation reservoir 129
proximal magnet
167b, for further processing (e.g., aspiration and delivery for amplification,
etc.).
[00118] As shown in FIGURE iiH, the separation subsystem 160 can provide a
seventh
operation mode 164g, wherein, in the seventh operation mode 164g, the support
structure
240' is coupled with a pipette interface or actuator interface 6 (e.g.,
described in relation to
the gantry 170 and pipettor 174) and the magnet 230' is coupled with the
adaptor 210' in the
retained position at the separation reservoir 129. In the seventh operation
mode 164g, the
adaptor 210', still coupled with the support structure, is retained in
position at the sleeve
stripping tool 165, and the adaptor 210' (with magnetically bound
functionalized particles) is
submerged within the fluid in the separation reservoir 129. In the seventh
operation mode
164g, magnet 167b is displaced toward the separation reservoir 129 (e.g., by
magnet actuator
169 described above) to prepare for attraction and retention of target or non-
target material
coupled to the functionalized particles of the fluid against a wall 128a of
the separation
reservoir 129. As such, the seventh operation mode 164g prepares target
material for transfer
from the adaptor 210' to a region of the separation reservoir 129 proximal
magnet 167b, for
further processing (e.g., aspiration and delivery for amplification, etc.).
[00119] As shown in FIGURE ill, the separation subsystem 160 can provide
an eighth
operation mode 164h, in which the support structure 240' is coupled with a
pipette interface
or actuator interface 6 (e.g., described in relation to the gantry 170 and
pipettor 174), and
moved away from the separation reservoir 129 to be replaced with a suitable
tip from the tool
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
container described above. In the eighth operation mode 164h, the magnet 230'
of is
uncoupled from the adaptor 210' above the separation reservoir 129, by having
the pipetting
head move the support structure 240' away from the adaptor 210' while the
adaptor 210' is
retained in position at the sleeve stripping tool 165. In the eighth operation
mode 164h, the
adaptor 210' is submerged within the fluid in the separation reservoir 129. In
the eighth
operation mode 164h, magnet 167b is still positioned in proximity to the
separation reservoir
129 (e.g., by magnet actuator 169 described above) for retention of target or
non-target
material coupled to functionalized particles of the fluid against a wall 128a
of the separation
reservoir 129. In the eighth operation mode 164h, the magnet 167b draws target
material
toward the bottom of the separation reservoir 129 (e.g., for later extraction
from the bottom
by the pipettor, for retention while the pipettor draws material unbound by
the magnet 167b).
As such, the eighth operation mode 164h allows material captured at the
adaptor 210' to be
transmitted and temporarily retained at a region of the separation container
129 for further
processing.
[00120] As shown in FIGURE 11J, the separation subsystem 160 can provide a
ninth
operation mode 164i, wherein, in the ninth operation mode 164i, the pipetting
head/actuator
interface 6 is coupled with a suitable tip and moved into the separation
reservoir 129 to
aspirate material from the separation reservoir 129. In the ninth operation
mode 164i, the
adaptor 210' is still retained in position above the separation reservoir 129
at the sleeve
stripping tool 165 and submerged within the fluid in the separation reservoir
129. In the ninth
operation mode 164i, magnet 167b is moved away from the separation reservoir
129 (e.g., by
magnet actuator 169 described above) in coordination with aspiration of
material by the
pipetting head. As such, the ninth operation mode 164i allows material
captured at the
adaptor 210' to be transmitted and temporarily retained at a region of the
separation
container 129 for further processing.
[00121] Variations of steps shown in FIGURES 11A-11J, related to target
material
retrieval and downstream processing can, however, be implemented using
variations of
system 200 described above, without involvement of the gantry 170 and/or the
interface of
pipettor 174.
36
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00122] FIGURES 12A through 12D depict additional views of configurations
of a
magnetic sleeve 1410 with respect to sleeve stripping tool 165 of a separation
reservoir 129 of
a process container 20, in relation to operation modes described above.
[00123] Variations of the separation subsystem 160 can, however, include
elements and
provide modes of operation for target material retrieval based upon one or
more of:
gravitational forces, buoyant forces, centrifugal forces, chemical separation,
and/or any other
suitable separation approaches. In yet another embodiment, target material
retrieval
operation by the separation subsystem 160 may be used to transfer target
particles from the
microwell chip to another substrate or another new empty microwell chip while
keeping the
relative spatial locations of the different particles being transferred.
2.6 System ¨ Embodiments for Retrieval by Gravity-Associated Forces
[00124] As shown in FIGURES 13A-13C, a variation of the system 300
includes an
adaptor 310 including a first region 311 configured to couple to a capture
region of a sample
processing cartridge 130 for capturing particles in single-particle format, a
second region 312,
and an internal cavity 320 passing from the first region 311 to the second
region 312; and a
support structure 340 reversibly coupled to the second region of the adaptor
310. The adaptor
310 can also include a vent 318 operable to prevent retention of an air bubble
between the
internal cavity 320 of the adaptor 310 and the capture region of the sample
processing chip
132.
[00125] The system 300 can also include one or more of: a set of plugs 350
configured
to couple an inlet and/or an outlet fluidly coupled to the capture region of
the sample
processing chip 132 (e.g., directly, or through a manifold device); and a
guide 360 including
a recess complementary to the sample processing chip 132 and the adaptor 310,
where the
guide 360 is configured to retain the sample processing chip 132 and coupled
adaptor 310
within a centrifuge apparatus for application of a gravity-associated force,
through
centrifugation, to contents of the capture region of the sample processing
chip 132 and to the
adaptor 310. The guide 360 can also function to prevent physical contact
between a centrifuge
apparatus and the sample processing chip 132 during operation.
[00126] The system 300 functions to allow an applied gravity-associated
force to the
capture region of the sample processing chip 132, as shown in FIGURE 13B, in
order to
provide a directional force for delivering target material within the capture
region, into the
37
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
adaptor 310. Embodiments of methods implemented with the system 300 can
produce
retrieval of target material in 2-3 minutes of manual operation time (and ¨15
minutes total
time), with a retrieval efficiency of ¨85-95%. The system 300 can thus
function to provide a
rapid method (in relation to manual operation time and total operation time)
with high
retrieval efficiency. The system 300 can implement one or more embodiments,
variations, or
examples of the method(s) described below, and/or can be used to implement
other methods.
2.6.1 Adaptor
[00127] As shown in FIGURES 13A-13C, the adaptor 310 includes a first
region 311
configured to couple to a capture region of a sample processing cartridge 130
for capturing
particles in single-particle format, a second region 312, and an internal
cavity 320 passing
from the first region to the second region. The adaptor 310 functions to
provide structures for
allowing target material to be delivered from the capture region of the sample
processing
cartridge 130 in a manner that promotes easy retrieval of the target material
from the adaptor
310, and to support application of a gravity-associated force to the capture
region for transfer
of target material of the sample processing chip 132 into the adaptor 310. The
adaptor 310
can also function to prevent air bubbles and/or other obstructions from
forming obstacles
between the capture region of the sample processing cartridge i3oand the
internal cavity 320
of the adaptor 310. The adaptor 310 can also function to prevent sample cross
contamination,
by serving as a disposable component that can be discarded between uses of the
system 300.
[00128] The adaptor 310 has an internal cavity 320 with a concave surface
facing
toward the capture region of the sample processing chip 132 (when the adaptor
310 is coupled
to the sample processing chip 132). The concave surface functions to define a
volume for
receiving and enabling force-based separation of target material from other
components
captured within the capture region of the chip 310. In variations, the volume
of the internal
cavity 320 can be from 0.1 microliters to 5mL; however, in alternative
variations, the volume
of the internal cavity can define another volume.
[00129] In variations, the surface of the internal cavity 320 can include
textures (e.g.,
dimples or other recesses, chambers, etc.), binding agents (e.g., chemical
agents, charged
agents, etc.) and/or other features that facilitate preferential retention of
target material at
the internal cavity 320 of the adaptor 320 after application of the applied
force.
38
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00130] As shown in FIGURE 13A, the adaptor 310 also includes a vent 318
configured
to allow air (or other gases) to be released from within the internal cavity
320, after the
adaptor 310 is coupled to the chip 310, in order to prevent air (or other
gases) from creating
a barrier to separation of target material from wells of the capture region of
the sample
processing chip 132, with application of the applied force. The vent 318 can
be placed at a
peripheral region of the adaptor 310, or can alternatively be placed at
another suitable region
of the adaptor. In relation to the applied force, the vent can be placed in an
orientation that
prevents target material from leaving the internal cavity 320 and through the
vent 318;
however, vent 318 can alternatively be placed in another orientation. The
adaptor 310 can
also include multiple vents or other bubble-releasing features (e.g., valves)
and/or self-sealing
material sections through which a needle can be pierced and air bubble
extracted.
[00131] The adaptor 310 preferably has a wall thickness suitable for
magnitudes of force
used for separation of target material. In examples, the wall thickness can
range from 0.2 to
3 mm thick; however, in other examples, the wall thickness can have any other
suitable
thickness.
[00132] The adaptor 310 can additionally or alternatively include
structural features
that enable operation modes of the system 300. For instance, in relation to
coupling and
release of the adaptor 310 from the capture region of the sample processing
cartridge 130, the
adaptor 310 can include a protrusion 314 (e.g., tab) that can be used to
facilitate coupling and
uncoupling of the adaptor 310 from the sample processing chip 132.
[00133] As described above, the adaptor 310 couples, at a first region 311,
to an exposed
capture region of the sample processing cartridge 130, in order to form a
volume for
separation and retrieval of target material from the capture region with
application of gravity-
associated force. The adaptor 310 can include a seal at the first region 311,
in order prevent
material from leaking at interfaces between the sample processing chip 132 and
the adaptor
310. The seal can be a separate element or an element integrated with the
adaptor 310. The
adaptor 310 can, however, omit a seal at the first region 311. The adaptor 310
also couples, at
a second region 312, to the support structure 340, for retention of the
adaptor 310 in position
at the sample processing chip 132, and for reversible coupling and removal
from the support
structure 340 and the sample processing chip 132. Coupling of the adaptor 310
to other
system components can occur with one or more of: a press fit, a snap fit, a
compression fit, a
39
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
friction fit, a male-female coupling interface, a screw, another fastener, a
magnetic
mechanism, and any other suitable mechanism.
[00134] The adaptor 310 can be composed of a polymeric material (e.g.,
plastic,
elastomer) that can undergo elastic deformation in order to facilitate removal
of air or other
gases trapped within the adaptor 310. The adaptor 210 can additionally or
alternatively
include (e.g., include particles of) or be composed of another material (e.g.,
non-polymeric
material, metal, ceramic, etc.) that has functionality for promoting
separation of target
material from non-target material captured within the capture region of the
sample
processing chip 132. The adaptor 310 can additionally or alternatively be
composed of any
other suitable material.
2.6.2 Support Structure
[00135] As shown in FIGURES 13A and 13C, the support structure 340 is
reversibly
coupled to the second region 312 of the adaptor 310. The support structure 340
functions to
retain the assembly of the sample processing chip 132 and the adaptor 310 in
position, and
to transition between operation modes for coupling and uncoupling the sample
processing
chip 132, adaptor 310, and/or guide 360 (described in more detail below).
[00136] The support structure 340 can have a form factor for clamping the
assembly of
the sample processing cartridge 130 and the adaptor 310 together in a manner
that prevents
material from leaking at the interface between the sample processing cartridge
130 and the
adaptor 310. In one variation, the support structure 340 can thus have the
form of a clamshell,
where terminal opposing regions include clamping structures for clamping the
assembly of
the adaptor 310 and the sample processing chip 132 together. The support
structure 340 can
also have an opening that allows contents of the adaptor 310 and/or capture
region of the
sample processing chip 132 to be observed during processing.
[00137] The support structure 340 can be composed of one or more polymeric
materials
(e.g., plastics) that are sanitizable (e.g., autoclavable, resistant to damage
by ethanol, etc.)
between uses of the system 300. However, the support structure 340 can
alternatively be
composed of another suitable material. Furthermore, the support structure 340
can be a
disposable or non-disposable component of the system 300.
2.6.3 Set of Plugs and Guide
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00138] As shown in FIGURE 3A, the system 300 can also include a set of
plugs 350
configured to couple an inlet and/or an outlet fluidly coupled to the capture
region of the
sample processing chip 132 (e.g., directly, or through a manifold device). In
embodiments
where the sample processing chip 132 (described above) includes a manifold or
other
substrate for fluid delivery into and from the sample processing chip 132, the
set of plugs 350
can couple to the inlet(s) and outlet(s) of the manifold. Additionally or
alternatively, the set
of plugs 350 can couple directly to an inlet and/or outlet of the sample
processing chip 132.
Coupling of the set of plugs 350 to other system components can occur with one
or more of:
a press fit, a snap fit, a friction fit, a male-female coupling interface, a
screw, another fastener,
a magnetic mechanism, and any other suitable mechanism.
[00139] The set of plugs 350 can be composed of one or more polymeric
materials (e.g.,
plastics) that are elastomeric and/or sanitizable (e.g., autoclavable,
resistant to damage by
ethanol, etc.) between uses of the system 300. However, the set of plugs can
alternatively be
composed of another suitable material. Furthermore, the set of plugs 350 can
be a disposable
or non-disposable component of the system 300.
[00140] As shown in FIGURES 13A and 13C, the system 300 can also include a
guide
360 including a recess complementary to the sample processing chip 132 and the
adaptor 310,
where the guide 360 is configured to retain the sample processing chip 132 and
coupled
adaptor 310 within a centrifuge apparatus for application of a gravity-
associated force,
through centrifugation, to contents of the capture region of the sample
processing chip 132
and to the adaptor 310. The guide 360 can also function to prevent physical
contact between
a centrifuge apparatus and the sample processing chip 132 during operation.
The recess of
the guide 360 preferably allows the entire sample processing chip 132 and
adaptor 310
assembly to be seated within the recess; however, the recess of the guide 360
can alternatively
be configured to receive only a portion of the sample processing chip 132
and/or adaptor 310.
[00141] As shown in FIGURE 13C, the recess can include an extended region
not
contacting the sample processing chip 132 and/or adaptor 310, where the
extended region
facilitates placement and removal of the chip assembly within the recess by an
operator.
Coupling of the sample processing chip 132 and/or adaptor 310 assembly to the
recess of the
guide 360 can occur with one or more of: a press fit, a snap fit, a
compression fit, a friction
41
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
fit, a male-female coupling interface, a screw, another fastener, a magnetic
mechanism, and
any other suitable mechanism.
[00142] The guide 360 can be composed of one or more polymeric materials
(e.g.,
plastics) that are rigid and/or sanitizable (e.g., autoclavable, resistant to
damage by ethanol,
etc.) between uses of the system 300. However, the guide 360 can alternatively
be composed
of another suitable material. Furthermore, the set of plugs 350 can be a
disposable or non-
disposable component of the system 300.
2.7 System ¨ Conclusion
[00143] The system(s) described can additionally or alternatively include
other
components that facilitate target material retrieval from a capture region of
a chip. The
system(s) described can implement one or more embodiments, variations, and
examples of
the method(s) described below, or any other suitable method.
3. Method
[00144] As shown in FIGURE 14, embodiments of a method 400 for target
material
retrieval include: capturing a set of particles, in single-particle format, at
a set of wells
distributed across a substrate at a capture region 41o; supporting an
environment for
processing target material of the set of particles within the capture region,
according to a set
of operations 420; forming an assembly with an adaptor configured to interact
with (e.g.,
couple to) to the substrate 430; transmitting a force to the adaptor and the
capture region,
thereby releasing target material of the set of particles into the adaptor
440; and releasing
target material of the set of particles for capture by the adaptor 450.
[00145] Embodiments, variations, and examples of the method 400 function
to provide
mechanisms for efficient retrieval of target material from a high-density
capture device (e.g.,
microwell chip), where the high-density capture device includes a high-density
array of high-
aspect ratio microwells, in order to promote increased efficiency in captured
single cell-bead
pairing efficiency. Embodiments of the method 400 can also function to reduce
manual
burden in relation to retrieval of target material from the high-density
capture device.
Embodiments of the method 400 can also function to increase the efficiency at
which target
material is retrieved from the high-density capture device, and the efficiency
at which non-
target material is retained at the capture device.
42
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00146] The method 400 can process target material from cells captured in
single cell
format at a capture region of a chip, as described above. The cells can
include any or all of
mammalian cells (e.g., human cells, mouse cells, etc.), embryos, stem cells,
plant cells,
microbes or any other suitable kind of cells. The target material can include
material
associated with the cells, tissue, nuclei or cell-free nucleic acids (e.g.,
target ysate, mRNA,
RNA, DNA, proteins, glycans, metabolites etc.) or particles bound with
cellular or cell-free
biomarkers. Additionally or alternatively, the method 400 can be configured to
process
particles (e.g., beads, probes, nucleotides, oligonucleotides,
polynucleotides, etc.), reagents,
or any other suitable materials as target materials for further processing.
The method also
can be configured to selectively remove multiple target particles
simultaneously from a
surface seeded with multitude of particles by selectively binding the target
particles with other
carrier particles that can be carried to another position by moving the
carrier particles with a
mechanism that moves the carrier particles.
[00147] The method 400 can be implemented by embodiments of the systems
described above, and/or any other suitable system components.
4.1 Method ¨ Capture and Processing Target Material
[00148] Block 410 recites: capturing a set of particles, in single-
particle format, at a set
of wells distributed across a substrate at a capture region. Block 410
functions to process
content of a sample in order to isolate particles (e.g., single cells, cells
co-captured with
functional particles, etc.) in single-particle format within individual
capture chambers of a
chip, in order to isolate target material from individual target particles in
a manner that
facilitates further downstream processing. Block 410 can be implemented by an
embodiment,
variation, or example of the sample processing cartridge 13o/sample processing
chip 132
described above; however, Block 410 can additionally or alternatively include
receiving a
biological sample at any other suitable system configured to capture cells in
at least one of
single-cell format and single-cluster format (e.g., with co-capture of cell in
single-cell format
and one or more functional particles corresponding to each single cell).
[00149] In Block 410, a biological sample containing the target particles
can be
transmitted and/or received directly into an inlet of the chip (e.g., by
pipetting, by fluid
delivery through a fluid channel coupled to the array) for distribution across
a set of wells of
a capture region of the chip, and/or in any other suitable manner.
Embodiments, variations,
43
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
and examples of Block 410 can be implemented as described in one or more
applications
incorporated by reference above.
[00150] Block 420 recites: supporting an environment for processing target
material of
the set of particles within the capture region, according to a set of
operations. Block 420
functions to create an environment whereby target material of the sample can
be prepared
for retrieval in coordination with application of an applied force, according
to subsequent
blocks of the method 400. As such, Block 420 can include creating physical
environments
(e.g., within chambers, with appropriate process reagents) for one or more of:
ysing captured
cells, disrupting membranes of captured cells; releasing target material
(e.g., nucleic acid
content) from captured cells; separating undesired elements (e.g., RNA,
proteins) captured
sample material; performing washing steps, co-capturing functional particles
(e.g., non-
magnetic beads, magnetic beads) with individually-captured cells and/or their
target
material; performing barcoding steps; attaching relevant adaptor molecules to
released
nucleic acid content; hybridizing target material (e.g., mRNA) to functional
particles;
performing reverse transcription; transmitting a retrieval buffer into the
chip for preparation
of target material for release from the capture region; sonicating or
otherwise physically
disturbing contents of the capture region for the chip for preparation of
target material for
release from the capture region; and/or performing any other suitable steps to
enable efficient
retrieval of target material from the capture region of the chip. As described
in more detail
below, specific steps for implementing magnetic force retrieval modes and/or
gravity-
associated retrieval modes can be performed.
[00151] Additionally or alternatively, embodiments, variations, and
examples of Block
420 can be implemented as described in one or more applications incorporated
by reference
above.
4.2 Method ¨ Magnetic Force Retrieval Modes
[00152] Related to embodiments, variations, and examples of the systems
200, 200'
described above, the method 400 can include steps for retrieval of target
material from the
capture region of the sample processing chip, using magnetic force retrieval
modes.
[00153] In particular, Block 430 recites: forming an assembly with an
adaptor
configured to couple to the substrate. Block 430 is preferably implemented by
way of an
embodiment, variation, or example of the adaptor 210, 210' described above,
whereby the
44
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
adaptor includes functionality for separating a magnet from physically
contacting wells or
other sensitive material at the capture region of the chip, and for
transmitting forces
associated with a magnetic field to the capture region for retrieval of target
material of the
chip. Forming the assembly can be facilitated through structural features of
the chip and/or
adaptor, as well as through use of guides or other support structures for
retaining relative
orientations between the chip and the adaptor during the process of delivering
captured
target material from the capture region of the chip for retrieval.
[00154] Block 440 recites: transmitting a force to the adaptor and the
capture region,
thereby releasing target material of the set of particles toward the adaptor.
Block 440
functions to transmit force, in a controlled manner, to the capture region of
the chip using the
adaptor, in order to promote release of target material from the chip for
retrieval. In relation
to magnetic retrieval modes, the force is a magnetic force generated through
use of a magnet
(e.g., such as the magnets described above); however, the force can
additionally or
alternatively include another suitable force. Furthermore, the force is
preferably applied as a
pulling force in a direction perpendicular to a plane at which the set of
wells is defined;
however, the force can alternatively be oriented in any other suitable
direction.
[00155] Block 450 recites: releasing target material of the set of
particles for capture by
the adaptor. Block 450 functions to promote transmission of target material
(e.g., through
use of coupled magnetic beads to functional particles to which target material
is bound)
toward the adaptor, in order to facilitate retrieval of target material from
the chip in an
efficient manner. Target material can then be extracted for further downstream
processing.
[00156] Variations and examples of magnetic retrieval modes in association
with Blocks
420-450 are further described in Section 4.2.1. below.
4.2.1 Magnetic Retrieval Method Variations and Examples
[00157] In particular, as shown in FIGURES 15A and 15B, variations of
magnetic
retrieval methods 500 can include steps for: Co-capturing single cells and
barcoded beads
within individual wells of the chip 502; Lysing cells and transferring mRNA
from cells to its
co-captured bead 504; linking captured mRNA/proteins to barcoded
oligonucleotide
sequence on beads through Reverse Transcriptase or ligation (where, during
this process,
only biotinylated TS0 primer attaches to bead in a microwell that has a target
cell) 506; thus
binding biomarkers from single cells to functionalized non-magnetic beads, in
single bead
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
format within a set of wells of the chip 51o; binding a set of magnetic
particles (e.g.,o.5-3
micron particles) through specific interactions to the functionalized non-
magnetic beads
within the set of wells 520 (e.g., where biotinylayed magnetic beads, 0.5-3
micron, bind to
target beads through streptavidin interactions and remaining excess bead are
freely floating,
laying on the floor or non-specifically bind to other beads); and using an
adaptor, applying a
magnetic force to the set of magnetic particles coupled to the captured target
material (e.g.,
directly coupled to functionalized non-magnetic microspheres within the wells,
directly
coupled to target material using a molecular scissor process), for retrieval
of target material
from within the set of wells 530. Magnetic retrieval methods described can
produce retrieval
of target material in 5-8 minutes of manual operation time (and 15-45 minutes
total time),
with a retrieval efficiency of> 90% where only magnetic particles coupled to
target material
of the sample are retrieved. Furthermore, magnetic retrieval methods can
produce a
reduction in reverse transcription-derived concatamers, provide an automation-
friendly
protocol, reduce number of splits required in downstream cDNA amplification,
reduce the
need for SPRI-based clean-up and size selection and reduce efforts and process
reagent usage
in downstream steps (e.g., associated with exonuclease treatment and cDNA
amplification).
Removal of exonuclease treatment steps in library preparation from single
cells is very
advantageous as any contamination of exonuclease enzymes to the instruments,
lab bench
and equipment used during one single cell preparation may inhibit the
reactions used for
subsequent single cell preparation for the next sample using the exonuclease
contaminated
elements.
[00158] First Example: In a first example of the methods 400 and 500,
magnetic forces
can be transmitted to the chip, for enabling a mechanism for binding and
selective removal
of barcoded non-magnetic microspheres coupled to or otherwise containing mRNA
products
as target material from single cells originally captured at the chip. In more
detail, as shown
in FIGURE 16, the method 600 can include functionality for: co-capturing
single cells and
single barcoded beads using different partitions (microwells, droplets) 610;
ysing cells in
partitions and transfer biomarkers to barcoded bead through binding
interactions 620;
performing linking of biomarkers to barcoded oligo tags on bead through
molecular reactions
(Reverse transcription, ligation etc.), where during molecular linking
reaction, biotinylated
primers get added to the bead containing target biomarkers only and these
linking reactions
46
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
may be done in the partitions or after partitions removed 630; washing off
unbound biotinylated primers 640; adding streptavidin coated magnetic beads
650;
optionally cutting off oligonucleotide sequence from beads using cleaving
mechanisms (e.g.,
molecular scissors, photocleaving, thermal cleaving, etc.), where cleaved
products having
target biomarkers bind to magnetic beads using biotin-streptavidin
interactions 660; and
using magnetic force to separate the target oligonucleotide complexed from the
remaining
oligonucleotide tags 670. Embodiments, variations, and examples of workflow
aspects can be
performed as described in relation to applications incorporated by reference
above.
[00159]
As shown in FIGURES. 17A-17D, an example of the method 700 can include
capturing functionalized non-magnetic beads in single-bead format 710, and
hybridizing the
functionalized non-magnetic beads to mRNA material from single cells captured
contemporaneously in corresponding wells 720. As shown in FIGURE 17A (top),
hybridization can involve use of a molecule having a set of thymine (T) bases
bound to the
functionalized non-magnetic bead, a template switching oligonucleotide (TSO)
sequence at a
5' end, a barcode sequence, a unique molecular identifier (UMI), and a tail at
the 3' end.
[00160]
Then, as shown in FIGURE 17A (bottom) the first example of the method 600
can include performing a reverse transcription reaction (RT-reaction) with the
target mRNA
material, with a first molecule corresponding to the target mRNA molecule and
having 3' end
non-templated cytosine (C) bases 730.
The RT-reaction thus incorporates a TSO
containing a biotin tag at the 5' end. Following Nock 730, the method 700 can
include
washing steps for removing unbound biotinylated TSO primers, as described in
applications
incorporated by reference above.
[00161]
Then, as shown in FIGURE 17B, the first example of the method 600 can
include implementing cDNA with a template switching oligonucleotide (TSO)
sequence
extension at the first strand having 3' end non-templated cytosine bases, for
complete capture
of 5' ends of the target material, using the template switching mechanism of a
Moloney
Murine Leukemia Virus (MMLV) RT enzyme 740. The cDNA TSO sequence extension
corresponds with a Biotin TSO at the 5' end of the target mRNA.
[00162]
Then as shown in FIGURES 17B and 17C, the first example of the method 700
can include binding 750 the functionalized non-magnetic beads to magnetic
streptavidin
beads (e.g., dynabeads) at the 5' biotin end of the molecule produced in Block
740, by way of
47
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
the biotyntilated TS0 portion. In more detail, the magnetic streptavidin beads
can be added
to the capture chambers and mixed with contents with an incubation period
(e.g., 20 minutes)
before retrieval. As shown in FIGURE 17C, the method 700 can further include
applying an
attractive magnetic force 660 to draw the bead complexes to the adaptor using
an
embodiment, variation, or example of the system 200 described above. As shown
in FIGURE
17D, captured bead complexes can then be transmitted to a container (e.g.,
tube) for further
processing.
[00163] Second Example: In a second example of the methods 400 and 500,
magnetic
forces can be transmitted to the chip, for enabling a mechanism for binding
and selective
removal of barcoded nucleic acid material products as target material from
single cells
originally captured at the chip, while leaving functionalized non-magnetic
particles at the chip
during the retrieval process. In more detail, as shown in FIGURES 18A-18E, an
example of
the method 800 can include capturing functionalized non-magnetic beads in
single-bead
format, and hybridizing the functionalized non-magnetic beads to mRNA material
from
single cells captured contemporaneously in corresponding wells. Then, instead
of retrieving
functionalized non-magnetic beads from the chambers of the chip, the method
800 can
implement molecular scissors (e.g., double stranded molecular scissor
molecules, single
stranded molecular scissor molecules) or photocleaving to release target cDNA-
RNA hybrid
for capture on secondary magnetic particles, for retrieval by applied magnetic
force.
Examples of molecular scissors include Btu Endonucleases that can specifically
cut single
started oligonucleotide sequence containing modified bases (e.g.,
deoxylUridine, dSpacer or
deoxylnosine. Another example of molecular scissors include Urasil-specific
Excision
Reagent (USER) enzymes that can specifically cut single stranded
oligonucleotide sequence
containing modified base Uracil. In examples, the enzyme is activated at a
temperature of
¨37C. Examples of photocleavable linkers (PC Linker) include a non-nucleosidic
moiety that
can be used to link two nucleotide sequences through a short UV photocleavable
C3 spacer
arm. Photo-cleavage of PC Linker by UV light yields one s'-phosphorylated
oligo and one 3'-
phosphorylated oligo. This process can select for molecules containing
products and reduce
damage to target cDNA-RNA hybrids due to reduced stresses attributed to
pulling a single
bead type from a capture well, as opposed to pulling two bead types out of the
wells).
48
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00164] In more detail, as shown in FIGURE 18A (top), hybridization can
involve use
of a molecule having a set of thymine (T) bases (e.g., 5 or 10 T bases) bound
to the
functionalized non-magnetic bead, a modified base region having a set of
modified, non-
natural bases (e.g. dU as shown in FIGURE 18A, top; dSpacer as shown in FIGURE
18A,
bottom) configured to be targeted by a molecular scissor (e.g., that operates
with a or
ultraviolet photocleaving mechanism, that operates with another mechanism),
template
switching oligonucleotide (TSO) sequence, a barcode sequence, a unique
molecular identifier
(UMI), and a tail at the 3' end.
[00165] The second example of the method 700 can include Blocks 830 and
840 (shown
in FIGURES 18B and 18C), analogous to Blocks 730 and 740 described above, for
performing
a RT reaction and a TS0 process for complete capture of 5' ends of target
material using an
MMLV RT enzyme.
[00166] Then, as shown in FIGURE 18C, the second example of the method 800
can
include releasing the target RNA-cDNA hybrids into the wells of the capture
region of the chip
85o by restricting the bead-oligonucleotides at the modified base region,
using single or
double stranded molecular scissors.
[00167] Then, as shown in FIGURE 18C and 18D, the second example of the
method
800 can include binding released target RNA-cDNA hybrids to magnetic
streptavidin beads
at the 5' biotin end of the molecule produces in Block 840. As shown in FIGURE
18D, the
method 800 can further include applying an attractive magnetic force 860 to
draw the bead
complexes to the adaptor using an embodiment, variation, or example of the
system 200
described above. As shown in FIGURE 18E, captured bead complexes can then be
transmitted
to a container (e.g., tube) for further processing.
[00168] While examples are described above, any other suitable target
material (e.g.,
non-mRNA material) can be processed using other enzymes (e.g., non-MMLV
enzymes),
other transcription processes, and/or any other suitable processes.
4.3 Method ¨ Gravity-Associated Force Retrieval Modes
[00169] Related to embodiments, variations, and examples of the system 300
described
above, the method 400 can include steps for retrieval of target material from
the capture
region of the chip, using gravity-associated force retrieval modes.
49
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
[00170] In particular, Block 430 recites: forming an assembly with an
adaptor
configured to couple to the substrate. Block 430 is preferably implemented by
way of an
embodiment, variation, or example of the adaptor described above, whereby the
adaptor
includes functionality for defining an internal cavity into which target
material can be
aggregated with application of an applied force to the assembly. Forming the
assembly can be
facilitated through structural features of the chip and/or adaptor, as well as
through use of
guides or other support structures for retaining relative orientations between
the chip and the
adaptor during the process of delivering captured target material from the
capture region of
the chip for retrieval.
[00171] Block 440 recites: transmitting a force to the adaptor and the
capture region,
thereby releasing target material of the set of particles into the adaptor.
Block 440 functions
to transmit force, in a controlled manner, to the capture region of the chip,
in order to
promote release of target material from the chip and into the adaptor for
retrieval. In relation
to gravity-associated force retrieval modes, the force is a centrifugal force
generated through
use of a centrifuge apparatus; however, the force can additionally or
alternatively include
another suitable force.
[00172] Block 450 recites: releasing target material of the set of
particles into the
adaptor. Block 450 functions to promote transmission of target material toward
the adaptor,
in order to facilitate retrieval of target material from the adaptor in an
efficient manner.
Target material can then be extracted for further downstream processing.
[00173] Variations and examples of centrifugation-associated retrieval
modes in
association with Blocks 420-450 are further described in Section 4.3.1. below.
4.3.1 Centrifugation-Associated Retrieval Method Variations and Examples
[00174] In particular, as shown in FIGURE 19, variations of centrifugation
retrieval
methods 900 can include steps for: receiving a retrieval buffer at the capture
region of the
chip 910 (e.g., with filling of the wells and blocking inlet and outlet ports
of the sample
processing chip with plugs); forming an assembly with an adaptor having a vent
for removal
of trapped air within an internal cavity defined between the adaptor and the
capture region
920 (e.g., with attachment of the adaptor and applying pressure to remove any
trapped air);
sonicating the assembly (e.g., at 47 kHz, at another frequency) to promote
detachment of
target material from surfaces of wells of the capture region 930; coupling the
assembly to a
CA 03138949 2021-11-01
WO 2020/263700 PCT/US2020/038647
support structure and to a guide for positioning the assembly within a
centrifuge apparatus
940; centrifuging the assembly (e.g., at moo relative centrifugal field) such
that applied
forces direct target material into the adaptor 950; and retrieving a volume
(e.g., pellet) of
separated target material from the adaptor 960 (e.g., using retrieval buffer
delivered through
a fluidic network of the sample processing chip).
[00175] Centrifugation-based retrieval methods can rapidly produce
retrieval of target
material in 2-3 minutes of manual operation time (and ¨15 minutes total time),
with a
retrieval efficiency of ¨85-95%.
5. Conclusion
[00176] The FIGURES illustrate the architecture, functionality and
operation of
possible implementations of systems, methods and computer program products
according to
preferred embodiments, example configurations, and variations thereof. In this
regard, each
block in the flowchart or block diagrams may represent a module, segment, or
portion of code,
which comprises one or more executable instructions for implementing the
specified logical
function(s). It should also be noted that, in some alternative
implementations, the functions
noted in the block can occur out of the order noted in the FIGURES. For
example, two blocks
shown in succession may, in fact, be executed substantially concurrently, or
the blocks may
sometimes be executed in the reverse order, depending upon the functionality
involved. It
will also be noted that each block of the Nock diagrams and/or flowchart
illustration, and
combinations of blocks in the Nock diagrams and/or flowchart illustration, can
be
implemented by special purpose hardware-based systems that perform the
specified
functions or acts, or combinations of special purpose hardware and computer
instructions.
[00177] As a person skilled in the art will recognize from the previous
detailed
description and from the figures and claims, modifications and changes can be
made to the
preferred embodiments of the invention without departing from the scope of
this invention
defined in the following claims.
51
